Anti-cd20 antibodies and methods of use

ABSTRACT

Compositions and methods are provided for treating diseases associated with CD20, including lymphomas, autoimmune diseases, and transplant rejections. Compositions include anti-CD20 antibodies capable of binding to a human CD20 antigen located on the surface of a human CD20-expressing cell, wherein the antibody has increased complement-dependent cell-mediated cytotoxicity (CDC) that is achieved by having at least one optimized CDR engineered within the variable region of the antibody. Compositions also include antigen-binding fragments, variants, and derivatives of the monoclonal antibodies, cell lines producing these antibody compositions, and isolated nucleic acid molecules encoding the amino acid sequences of the antibodies. The invention further includes pharmaceutical compositions comprising the anti-CD20 antibodies of the invention, or antigen-binding fragments, variants, or derivatives thereof, in a pharmaceutically acceptable carrier, and methods of use of these anti-CD20 antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/850,604, filed Oct. 10, 2006, herein incorporated by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named332362SEQLIST.txt, created on Aug. 9, 2007, and having a size of 21.8kilobytes and is filed concurrently with the specification. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to antibodies capable of binding to CD20, methodsof using the antibodies, and methods for treatment of diseasesassociated with CD20-expressing cells.

BACKGROUND OF THE INVENTION

The CD20 molecule (also called human B-lymphocyte-restricteddifferentiation antigen or Bp35) is a hydrophobic transmembrane proteinwith a molecular weight of approximately 35 kD located on pre-B andmature B lymphocytes (Valentine et al. (1989) J. Biol. Chem.264(19):11282-11287; and Einfield et al. (1988) EMBO J. 7(3):311-717).CD20 is found on the surface of greater than 90% of B cells fromperipheral blood or lymphoid organs and is expressed during early pre-Bcell development and remains until plasma cell differentiation. CD20 ispresent on both normal B cells as well as malignant B cells. Inparticular, CD20 is expressed on greater than 90% of B cellnon-Hodgkin's lymphomas (NHL) (Anderson et al. (1984) Blood63(6):1424-1433), but is not found on hematopoietic stem cells, pro-Bcells, normal plasma cells, or other normal tissue (Tedder et al. (1985)J. Immunol. 135(2):973-979).

The 85 amino acid carboxyl-terminal region of the CD20 protein islocated within the cytoplasm. The length of this region contrasts withthat of other B cell-specific surface structures such as IgM, IgD, andIgG heavy chains or histocompatibility antigens class II α or β chains,which have relatively short intracytoplasmic regions of 3, 3, 28, 15,and 16 amino acids, respectively (Komaromy et al. (1983) NAR11:6775-6785). Of the last 61 carboxyl-terminal amino acids, 21 areacidic residues, whereas only 2 are basic, indicating that this regionhas a strong net negative charge. The GenBank Accession No. isNP_(—)690605.

It is thought that CD20 might be involved in regulating an early step(s)in the activation and differentiation process of B cells (Tedder et al.(1986) Eur. J. Immunol. 16:881-887) and could function as a calcium ionchannel (Tedder et al. (1990) J. Cell. Biochem. 14D:195).

Despite uncertainty about the actual function of CD20 in promotingproliferation and/or differentiation of B cells, it provides animportant target for antibody-mediated therapy to control or kill Bcells involved in cancers and autoimmune disorders. In particular, theexpression of CD20 on tumor cells, e.g., NHL, makes it an importanttarget for antibody-mediated therapy to specifically target therapeuticagents against CD20-positive neoplastic cells. However, while theresults obtained to date clearly establish CD20 as a useful target forimmunotherapy, they also show that currently available murine andchimeric antibodies do not constitute ideal therapeutic agents.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods are provided for treating diseases associatedwith CD20, including lymphomas, autoimmune diseases, and transplantrejections. Compositions include anti-CD20 antibodies capable of bindingto a human CD20 antigen located on the surface of a humanCD20-expressing cell, wherein the antibody has increasedcomplement-dependent cell-mediated cytotoxicity (CDC) in comparison torituximab. Compositions also include antigen-binding fragments,variants, and derivatives of the monoclonal antibodies, cell linesproducing these antibody compositions, and isolated nucleic acidmolecules encoding the amino acid sequences of the antibodies. Theinvention further includes pharmaceutical compositions comprising theanti-CD20 antibodies of the invention, or antigen-binding fragments,variants, or derivatives thereof, in a pharmaceutically acceptablecarrier.

The monoclonal antibodies disclosed herein have a strong affinity forCD20 and are characterized by improved CDC function in comparison torituximab. The antibodies of the invention mediate killing of the cellsexpressing CD20. The antibodies of the invention are capable ofspecifically binding to a human CD20 antigen expressed on the surface ofa human cell and are characterized by having at least one optimizedcomplementarity-determining region (CDR) in the anti-CD20 heavy chainand/or the anti-CD20 light chain. The optimized CDR has been modifiedand comprises a sequence set forth in any one of SEQ ID NOS:1-8.Particular antibody sequences are disclosed having changes in at leastone CDR of the anti-CD20 heavy chain, and in the CDR3 of the lightchain. Compositions of the invention comprise anti-CD20 antibodies, andantigen-binding antibody fragments, variants, and derivatives thereof,comprising at least one optimized CDR.

In one embodiment of the invention, methods of treatment compriseadministering to a patient a therapeutically effective dose of apharmaceutical composition comprising suitable anti-CD20 antibodies, orantigen-binding fragments, variants, or derivatives thereof. Diseasesassociated with CD20-expressing cells include autoimmune diseases,cancers, and organ and tissue graft rejections. Lymphomas that can betreated or prevented by a method of the invention include non-Hodgkin'slymphomas (high-grade lymphomas, intermediate grade lymphomas, and lowgrade lymphomas), Hodgkin's disease, acute lymphoblastic leukemias,myelomas, chronic lymphocytic leukemias, and myeloblastic leukemias.

Particular autoimmune diseases contemplated for treatment using themethods of the invention include systemic lupus erythematosus (SLE),rheumatoid arthritis, Crohn's disease, psoriasis, autoimmunethrombocytopenic purpura, multiple sclerosis, ankylosing spondylitis,myasthenia gravis, and pemphigus vulgaris. Such antibodies may also beused to prevent rejection of organ and tissue grafts by suppressingautoimmune responses, to treat lymphomas by targeting and killingB-lymphocytes, and to deliver toxins to CD20-bearing cells in a specificmanner.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 sets forth the amino acid sequences for the H1286 variable heavydomain (SEQ ID NO:29) and L373 variable light domain (SEQ ID NO:10) forthe original humanized murine anti-CD20 monoclonal antibody (mAb 1097).The residues making up the complementarity-determining regions (CDR1,CDR2, and CDR3) for each variable domain are underlined.

FIG. 2 shows the substitutions made within CDR3 of the H1286 variableheavy domain of the humanized murine anti-CD20 mAb 1097 (this CDR3 isdesignated “271”) to produce the 1236 (SEQ ID NO:1), 1237 (SEQ ID NO:2),and 1238 (SEQ ID NO:3) optimized CDR3s (see Example 1). The 271 CDR3sequence (SEQ ID NO:30) is equivalent to that for the CDR3 of thevariable domain within the heavy chain of the chimeric murine/humananti-CD20 antibody known as C2B8 (rituximab).

FIG. 3 sets forth the amino acid sequences for the optimized H1569 (SEQID NO:12), H1570 (SEQ ID NO:13), and H1571 (SEQ ID NO:14) variable heavydomains. H1569 comprises the optimized 1236 CDR3 (SEQ ID NO:1); H1570comprises the optimized 1237 CDR3 (SEQ ID NO:2), and H1571 comprises theoptimized 1238 CDR3 (SEQ ID NO:3). Each of these optimized variableheavy domains was paired with the L373 variable light domain (SEQ IDNO:10) to produce optimized mAb 1236, mAb 1237, and mAb 1238. Theresidues making up CDR1, CDR2, and CDR3, respectively, for each variabledomain are underlined.

FIG. 4 sets forth the coding sequences for the optimized H1569 (SEQ IDNO:19), H1570 (SEQ ID NO:20), and H1571 (SEQ ID NO:21) variable heavydomains.

FIG. 5 sets forth the amino acid (SEQ ID NO:10) and coding (SEQ IDNO:23) sequences for the L373 variable light domain. The residues andcoding sequences for CDR1, CDR2, and CDR3, respectively, are underlined.

FIGS. 6A-6D show results of binding specificity for optimized humanizedmAb 1236, mAb1237, and mAb 1238 as compared to mAb 271 (having theidentical sequence to rituximab). Binding specificity of these optimizedmAbs for CHO cells expressing CD20 (FIG. 6A), CD20⁺ EB1 cells (FIG. 6B),CD20⁻ CHO vector (FIG. 6C), and CD20⁻ NSO (FIG. 6D) was assessed byFluorometric Microvolume Assay Technology (FMAT). See Example 2. NSO isa mouse myeloma cell line that does not express human CD20.

FIG. 7 shows results of a murine 2H7 blocking assay to determine epitoperecognition of optimized mAbs 1236, 1237, and 1238 as compared to mAb271. The optimized mAbs all recognize the same epitope as mAb 271. SeeExample 2.

FIG. 8 shows results of a CDC-mAb off-rate assay to determine complementdependent cytotoxicity (CDC) of optimized mAbs 1236, 1237, and 1238 ascompared to mAb 271. The optimized mAbs have increased CDC functionalactivity relative to that observed for mAb 271. See Example 2.

FIG. 9 shows results of an ADCC assay to determine antibody-dependentcellular cytotoxicity (ADCC) activity of optimized mAbs 1236, 1237, and1238 against Daudi target cells as compared to mAb 271. The optimizedmAbs have ADCC activity that is at least equivalent to that observed formAb 271. See Example 2.

FIG. 10 shows results of a direct apoptosis assay (independent of CDCand ADCC activity) to determine apoptosis activity of optimized mAbs1236, 1237, and 1238 against Ramos (NHL) cells as compared to mAb 271and mAb 11, which served as the isotype control. The optimized mAbs areas effective at inducing apoptosis as mAb 271. See Example 2.

FIG. 11 sets forth the amino acid sequences for the H1639 variable heavydomain (SEQ ID NO:16) and L373 variable light domain (SEQ ID NO:10) foroptimized mAb 1589. The residues making up thecomplementarity-determining regions (CDR1, CDR2, and CDR3) for eachvariable domain are underlined.

FIG. 12 sets forth the coding sequences for the H1639 variable heavydomain (SEQ ID NO:22) and L373 variable light domain (SEQ ID NO:23)within the heavy and light chains, respectively, of optimized mAb 1589.Codons for the CDR1, CDR2, and CDR3, respectively, of the variable heavyand variable light domains are underlined.

FIG. 13 shows results of a murine 2H7 blocking assay to determineepitope recognition of optimized mAbs 1588, 1589, 1590, 1652, and 1692as compared to mAb 271, negative control antibody mAb 11, optimized mAb1237, and rituximab (Rituxan®) With the exception of the negativecontrol, all tested mAbs recognize the identical or overlappingepitopes. See Example 4.

FIG. 14 shows results of a CDC assay to determine CDC activity ofoptimized mAbs 1588, 1589, 1590, 1652, and 1692 against target Daudi(NHL) cells as compared to that observed for mAb 271, 11, and optimizedmAb 1237. All optimized mAbs generally mediate greater CDC functionalactivity than does mAb 271. See Example 4.

FIGS. 15A and 15B show results of a CDC assay to determine CDC activityof optimized mAbs 1588, 1589, 1590, 1652, and 1692 against two targetB-CLL cell lines, EHEB cells (FIG. 15A) and MEC-1 cells (FIG. 15B), ascompared to that observed for mAb 271, mAb 11, and optimized mAb 1237.All optimized mAbs have increased CDC functional activity relative tomAb 271. See Example 4.

FIG. 16 shows results of a CDC-mAb off-rate assay to determine CDCactivity of optimized mAbs 1588, 1590, 1652, and 1692 as compared to mAb271 and optimized mAb 1237. All optimized mAbs have increased CDCfunctional activity relative to that observed for mAb 271. See Example4.

FIGS. 17A and 17B show results of a non-radioactive CDC assay todetermine CDC activity of optimized mAbs 1588, 1589, 1590, 1652, and1692 against two target NHL cell lines, Daudi cells (FIG. 17A) andWIL2-S cells (FIG. 17B), as compared to that observed for mAb 271,optimized mAb 1237, and negative control mAb 11 (designated “NEG” inthese figures). See Example 4.

FIGS. 18A and 18B show results of a non-radioactive CDC assay todetermine CDC activity of optimized mAbs 1588, 1589, 1590, 1652, and1692 against two target B-CLL cell lines, EHEB cells (FIG. 18A) andMEC-1 cells (FIG. 18B), as compared to that observed for mAb 271,optimized mAb 1237, and negative control mAb 11 (designated “NEG” inthese figures). See Example 4.

FIGS. 19A and 19B show results of a non-radioactive CDC assay todetermine CDC activity of optimized mAbs 1588, 1589, 1590, 1652, and1692 against two target EBV-transformed B cell lines (“normal” B cells),SS BLCL cells (FIG. 19A) and MelK BLCL cells (FIG. 19B), as compared tothat observed for mAb 271, optimized mAb 1237, and negative control mAb11 (designated “NEG” in these figures). See Example 4.

FIG. 20 shows results of an ADCC assay to determine ADCC activity ofoptimized mAbs 1588, 1589, 1590, 1652, and 1692 against Daudi (NHL)cells as compared to mAb 271, optimized mAb 1237, and control IgG. Theoptimized mAbs have ADCC activity that is similar to that observed formAb 271. See Example 4.

FIG. 21 shows results of an ADCC assay to determine ADCC activity ofoptimized mAbs 1588, 1589, 1590, 1652, and 1692 against MEC-1 (B-CLL)cells as compared to mAb 271, optimized mAb 1237, and control IgG. Theoptimized mAbs have ADCC activity that is similar to that observed formAb 271. See Example 4.

FIG. 22 shows a direct apoptosis assay (independent of CDC and ADCCactivity) to determine apoptosis activity of optimized mAbs 1236, 1237,and 1238 against Ramos (NHL) cells as compared to mAb 271 and mAb 11,which served as the isotype control. The optimized mAbs are as effectiveat inducing apoptosis as mAb 271. See Example 4.

FIG. 23 shows results of a whole-blood assay to determine cell killingactivity of optimized mAbs 1588, 1589, 1590, 1652, and 1692 againstDaudi (NHL) cells as compared to mAb 271 and optimized mAb 1237. Theoptimized mAbs have equivalent or better lysis against these cells ascompared to mAb 271. See Example 4.

FIGS. 24A and 24B show results of a whole-blood assay to determine cellkilling activity of optimized mAbs 1588, 1589, 1590, 1652, and 1692against two target B-CLL cell lines, EHEB cells (FIG. 24A) and MEC-1cells (FIG. 24B) as compared to mAb 271 and optimized mAb 1237. Theoptimized mAbs have equivalent or better lysis against these cells ascompared to mAb 271. See Example 4.

FIG. 25 shows results of an ADCC whole-blood assay to determine ADCCactivity of optimized mAbs 1588, 1589, 1590, 1652, and 1692 against theEBV-transformed B cell line MelK BLCL (“normal” B cells) as compared tomAb 271 and optimized mAb 1237. The optimized mAbs have equivalent orbetter lysis against these cells as compared to mAb 271. See Example 4.

FIG. 26 shows results of an in vivo test of optimized humanizedanti-mouse CD20 monoclonal antibody 1589 to determine the activity ofmAb 1589 in a Daudi cell xenograft model. The mAb 1589 antibody iseffective at prolonging the survival of mice with human Daudi cellxenografts. See Example 5.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an anti-CD20 antibody” is understood torepresent one or more anti-CD20 antibodies. As such, the terms “a” (or“an”), “one or more,” and “at least one” can be used interchangeablyherein.

As used herein, the term “tumor” refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerouscells and tissues.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto lymphoma and leukemia.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

A polypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides that do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid residue, e.g., a serine residue or an asparagineresidue.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purpose of the invention, as are native orrecombinant polypeptides that have been separated, fractionated, orpartially or substantially purified by any suitable technique.

Also included as polypeptides of the present invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. The terms “fragment,” “variant,” “derivative,” and“analog” when referring to anti-CD20 antibodies or antibody polypeptidesof the present invention include any polypeptides that retain at leastsome of the antigen-binding properties of the corresponding antibody orantibody polypeptide of the invention. Fragments of polypeptides of thepresent invention include proteolytic fragments, as well as deletionfragments, in addition to specific antibody fragments discussedelsewhere herein. Variants of anti-CD20 antibodies and antibodypolypeptides of the present invention include fragments as describedabove, and also polypeptides with altered amino acid sequences due toamino acid substitutions, deletions, or insertions. Variants may occurnaturally or be non-naturally occurring. Non-naturally occurringvariants may be produced using art-known mutagenesis techniques. Variantpolypeptides may comprise conservative or non-conservative amino acidsubstitutions, deletions, or additions. Derivatives of anti-CD20antibodies and antibody polypeptides of the present invention, arepolypeptides that have been altered so as to exhibit additional featuresnot found on the reference antibody or antibody polypeptide of theinvention. Examples include fusion proteins. Variant polypeptides mayalso be referred to herein as “polypeptide analogs.” As used herein a“derivative” of an anti-CD20 antibody or antibody polypeptide refers toa subject polypeptide having one or more residues chemically derivatizedby reaction of a functional side group. Also included as “derivatives”are those peptides that contain one or more naturally occurring aminoacid derivatives of the twenty standard amino acids. For example,4-hydroxyproline may be substituted for proline; 5-hydroxylysine may besubstituted for lysine; 3-methylhistidine may be substituted forhistidine; homoserine may be substituted for serine; and ornithine maybe substituted for lysine.

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA(pDNA). A polynucleotide may comprise a conventional phosphodiester bondor a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The term “nucleic acid” refers to any oneor more nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide. By “isolated” nucleic acid or polynucleotide is intendeda nucleic acid molecule, DNA or RNA, that has been removed from itsnative environment. For example, a recombinant polynucleotide encodingan anti-CD20 antibody contained in a vector is considered isolated forthe purposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, a polynucleotide or a nucleic acid may be or may include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator.

As used herein, a “coding region” is a portion of nucleic acid thatconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions, e.g., a single vector mayseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a nucleic acid encoding ananti-CD20 antibody or fragment, variant, or derivative thereof.Heterologous coding regions include without limitation specializedelements or motifs, such as a secretory signal peptide or a heterologousfunctional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid that encodesa polypeptide normally may include a promoter and/or other transcriptionor translation control elements operably associated with one or morecoding regions. An operable association is when a coding region for agene product, e.g., a polypeptide, is associated with one or moreregulatory sequences in such a way as to place expression of the geneproduct under the influence or control of the regulatory sequence(s).Two DNA fragments (such as a polypeptide coding region and a promoterassociated therewith) are “operably associated” if induction of promoterfunction results in the transcription of mRNA encoding the desired geneproduct and if the nature of the linkage between the two DNA fragmentsdoes not interfere with the ability of the expression regulatorysequences to direct the expression of the gene product or interfere withthe ability of the DNA template to be transcribed. Thus, a promoterregion would be operably associated with a nucleic acid encoding apolypeptide if the promoter was capable of effecting transcription ofthat nucleic acid. The promoter may be a cell-specific promoter thatdirects substantial transcription of the DNA only in predeterminedcells. Other transcription control elements, besides a promoter, forexample enhancers, operators, repressors, and transcription terminationsignals, can be operably associated with the polynucleotide to directcell-specific transcription. Suitable promoters and other transcriptioncontrol regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions that function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited to,ribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide of the present invention is RNA,for example, in the form of messenger RNA (mRNA).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions that encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal peptideor secretory leader sequence that is cleaved from the mature proteinonce export of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that polypeptides secreted by vertebrate cells generally have asignal peptide fused to the N-terminus of the polypeptide, which iscleaved from the complete or “full length” polypeptide to produce asecreted or “mature” form of the polypeptide. In certain embodiments,the native signal peptide, e.g., an immunoglobulin heavy chain or lightchain signal peptide is used, or a functional derivative of thatsequence that retains the ability to direct the secretion of thepolypeptide that is operably associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, may be used. For example, the wild-type leader sequence may besubstituted with the leader sequence of human tissue plasminogenactivator (TPA) or mouse β-glucuronidase.

The present invention is directed to certain anti-CD20 antibodies, orantigen-binding fragments, variants, or derivatives thereof. Unlessspecifically referring to full-sized antibodies such as naturallyoccurring antibodies, the term “anti-CD20 antibodies” encompassesfull-sized antibodies as well as antigen-binding fragments, variants,analogs, or derivatives of such antibodies, e.g., naturally occurringantibody or immunoglobulin molecules or engineered antibody molecules orfragments that bind antigen in a manner similar to antibody molecules.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin comprises at least the variabledomain of a heavy chain, and normally comprises at least the variabledomains of a heavy chain and a light chain. Basic immunoglobulinstructures in vertebrate systems are relatively well understood. See,e.g., Harlow et al. (1988) Antibodies: A Laboratory Manual (2nd ed.;Cold Spring Harbor Laboratory Press).

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or epsilon,(γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). It is thenature of this chain that determines the “class” of the antibody as IgG,IgM, IgA, IgD, or IgE, respectively. The immunoglobulin subclasses(isotypes) e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, etc., are wellcharacterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes are readilydiscernable to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of the instant invention.Although all immunoglobulin classes are clearly within the scope of thepresent invention, the following discussion will generally be directedto the IgG class of immunoglobulin molecules. With regard to IgG, astandard immunoglobulin molecule comprises two identical light chainpolypeptides of molecular weight approximately 23,000 Daltons, and twoidentical heavy chain polypeptides of molecular weight 53,000-70,000Daltons. The four chains are typically joined by disulfide bonds in a“Y” configuration wherein the light chains bracket the heavy chainsstarting at the mouth of the “Y” and continuing through the variableregion.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells, orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology referred to as the “constant region” and the“variable region.” The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (V_(L)) and heavy (V_(H)) chain portionsdetermine antigen recognition and specificity. Conversely, the constantdomains of the light chain (C_(L)) and the heavy chain (C_(H)1, C_(H)2,or C_(H)3) confer important biological properties such as secretion,transplacental mobility, Fc receptor binding, complement binding, andthe like. By convention the numbering of the constant region domainsincreases as they become more distal from the antigen binding site oramino-terminus of the antibody. The N-terminal portion is a variableregion and at the C-terminal portion is a constant region; the C_(H)3and C_(L) domains actually comprise the carboxy-terminus of the heavyand light chain, respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the V_(L) domain and V_(H) domain, or subset of the complementaritydetermining regions (CDRs) within these variable domains, of an antibodycombine to form the variable region that defines a three dimensionalantigen binding site. This quaternary antibody structure forms theantigen binding site present at the end of each arm of the Y. Morespecifically, the antigen binding site is defined by three CDRs on eachof the V_(H) and V_(L) chains. In some instances, e.g., certainimmunoglobulin molecules derived from camelid species or engineeredbased on camelid immunoglobulins, a complete immunoglobulin molecule mayconsist of heavy chains only, with no light chains. See, e.g.,Hamers-Casterman et al. (1993) Nature 363:446-448.

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen binding domain are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen binding domain as the antibody assumes its threedimensional configuration in an aqueous environment. The remainder ofthe amino acids in the antigen binding domains, referred to as“framework” regions, show less inter-molecular variability. Theframework regions largely adopt a β-sheet conformation and the CDRs formloops that connect, and in some cases form part of, the β-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable domainby one of ordinary skill in the art, since they have been preciselydefined (see, “Sequences of Proteins of Immunological Interest,” Kabatet al. (1983) U.S. Department of Health and Human Services; and Chothiaand Lesk (1987) J. Mol. Biol., 196:901-917, which are incorporatedherein by reference in their entireties).

In the case where there are two or more definitions of a term that isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al. (1983) U.S. Dept. of Health and HumanServices, “Sequences of Proteins of Immunological Interest” and byChothia and Lesk (1987) J. Mol. Biol. 196:901-917, which areincorporated herein by reference, where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of either definition to refer to a CDRof an antibody or variants thereof is intended to be within the scope ofthe term as defined and used herein. The appropriate amino acid residuesthat encompass the CDRs as defined by each of the above cited referencesare set forth below in Table I as a comparison. The exact residuenumbers that encompass a particular CDR will vary depending on thesequence and size of the CDR. Those skilled in the art can routinelydetermine which residues comprise a particular CDR given the variableregion amino acid sequence of the antibody. TABLE 1 CDR Definitions¹Kabat Chothia V_(H) CDR1 31-35 26-32 V_(H) CDR2 50-65 52-58 V_(H) CDR3 95-102  95-102 V_(L) CDR1 24-34 26-32 V_(L) CDR2 50-56 50-52 V_(L) CDR389-97 91-96¹Numbering of all CDR definitions in Table 1 is according to thenumbering conventions set forth by Kabat et al. (see below).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al. (1983) U.S. Dept. ofHealth and Human Services, “Sequence of Proteins of ImmunologicalInterest.” Unless otherwise specified, references to the numbering ofspecific amino acid residue positions in an anti-CD20 antibody orantigen-binding fragment, variant, or derivative thereof of the presentinvention are according to the Kabat numbering system.

In camelid species, the heavy chain variable region, referred to asV_(H)H, forms the entire antigen-binding domain. The main differencesbetween camelid V_(H)H variable regions and those derived fromconventional antibodies (V_(H)) include (a) more hydrophobic amino acidsin the light chain contact surface of V_(H) as compared to thecorresponding region in V_(H)H, (b) a longer CDR3 in V_(H)H, and (c) thefrequent occurrence of a disulfide bond between CDR1 and CDR3 in V_(H)H.

Antibodies or antigen-binding fragments, variants, or derivativesthereof of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized, primatized, or chimericantibodies, single chain antibodies, epitope-binding fragments, e.g.,Fab, Fab′ and F(ab′)₂, Fd, Fvs, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv), fragments comprising either aV_(L) or V_(H) domain, fragments produced by a Fab expression library,and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Idantibodies to anti-CD20 antibodies disclosed herein). ScFv molecules areknown in the art and are described, e.g., in U.S. Pat. No. 5,892,019.Immunoglobulin or antibody molecules of the invention can be of any type(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1, and IgA2, etc.), or subclass of immunoglobulin molecule.

Antibody fragments, including single-chain antibodies, may comprise thevariable region(s) alone or in combination with the entirety or aportion of the following: hinge region, C_(H)1, C_(H)2, and C_(H)3domains. Also included in the invention are antigen-binding fragmentsalso comprising any combination of variable region(s) with a hingeregion, C_(H)1, C_(H)2, and C_(H)3 domains. Antibodies or immunospecificfragments thereof for use in the diagnostic and therapeutic methodsdisclosed herein may be derived from any animal origin including birdsand mammals. Preferably, the antibodies are derived from human, murine,donkey, rabbit, goat, guinea pig, camel, llama, horse, or chickenantibodies. In another embodiment, the variable region may becondricthoid in origin (e.g., from sharks). As used herein, “human”antibodies include antibodies having the amino acid sequence of a humanimmunoglobulin and include antibodies isolated from human immunoglobulinlibraries or from animals transgenic for one or more humanimmunoglobulins and that do not express endogenous immunoglobulins, asdescribed infra and, for example in, U.S. Pat. No. 5,939,598 byKucherlapati et al. As used herein, the term “heavy chain portion”includes amino acid sequences derived from an immunoglobulin heavychain. A polypeptide comprising a heavy chain portion comprises at leastone of: a C_(H)1 domain, a hinge (e.g., upper, middle, and/or lowerhinge region) domain, a C_(H)2 domain, a C_(H)3 domain, or a variant orfragment thereof. For example, a binding polypeptide for use in theinvention may comprise a polypeptide chain comprising a C_(H)1 domain; apolypeptide chain comprising a C_(H)1 domain, at least a portion of ahinge domain, and a C_(H)2 domain; a polypeptide chain comprising aC_(H)1 domain and a C_(H)3 domain; a polypeptide chain comprising aC_(H)1 domain, at least a portion of a hinge domain, and a C_(H)3domain, or a polypeptide chain comprising a C_(H)1 domain, at least aportion of a hinge domain, a C_(H)2 domain, and a C_(H)3 domain. Inanother embodiment, a polypeptide of the invention comprises apolypeptide chain comprising a C_(H)3 domain. Further, a bindingpolypeptide for use in the invention may lack at least a portion of aC_(H)2 domain (e.g., all or part of a C_(H)2 domain). As set forthabove, it will be understood by one of ordinary skill in the art thatthese domains (e.g., the heavy chain portions) may be modified such thatthey vary in amino acid sequence from the naturally occurringimmunoglobulin molecule.

In certain anti-CD20 antibodies, or antigen-binding fragments, variants,or derivatives thereof disclosed herein, the heavy chain portions of onepolypeptide chain of a multimer are identical to those on a secondpolypeptide chain of the multimer. Alternatively, heavy chainportion-containing monomers of the invention are not identical. Forexample, each monomer may comprise a different target binding site,forming, for example, a bispecific antibody.

The heavy chain portions of a binding polypeptide for use in thediagnostic and treatment methods disclosed herein may be derived fromdifferent immunoglobulin molecules. For example, a heavy chain portionof a polypeptide may comprise a C_(H)1 domain derived from an IgG1molecule and a hinge region derived from an IgG3 molecule. In anotherexample, a heavy chain portion can comprise a hinge region derived, inpart, from an IgG1 molecule and, in part, from an IgG3 molecule. Inanother example, a heavy chain portion can comprise a chimeric hingederived, in part, from an IgG1 molecule and, in part, from an IgG4molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain. Preferably, thelight chain portion comprises at least one of a V_(L) or C_(L) domain.

Anti-CD20 antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein may be described or specified interms of the epitope(s) or portion(s) of an antigen, e.g., a targetpolypeptide (CD20) that they recognize or specifically bind. The portionof a target polypeptide that specifically interacts with the antigenbinding domain of an antibody is an “epitope,” or an “antigenicdeterminant.” A target polypeptide may comprise a single epitope, buttypically comprises at least two epitopes, and can include any number ofepitopes, depending on the size, conformation, and type of antigen.Furthermore, it should be noted that an “epitope” on a targetpolypeptide may be or include non-polypeptide elements, e.g., an epitopemay include a carbohydrate side chain.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes preferably contain at least seven, more preferably at leastnine and most preferably between at least about 15 to about 30 aminoacids. Since a CDR can recognize an antigenic peptide or polypeptide inits tertiary form, the amino acids comprising an epitope need not becontiguous, and in some cases, may not even be on the same peptidechain. In the present invention, peptide or polypeptide epitoperecognized by anti-CD20 antibodies of the present invention contains asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 15, at least20, at least 25, or between about 15 to about 30 contiguous ornon-contiguous amino acids of CD20.

By “specifically binds,” it is generally meant that an antibody binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, an antibody is said to “specifically bind”to an epitope when it binds to that epitope, via its antigen bindingdomain more readily than it would bind to a random, unrelated epitope.The term “specificity” is used herein to qualify the relative affinityby which a certain antibody binds to a certain epitope. For example,antibody “A” may be deemed to have a higher specificity for a givenepitope than antibody “B,” or antibody “A” may be said to bind toepitope “C” with a higher specificity than it has for related epitope“D.”

By “preferentially binds,” it is meant that the antibody specificallybinds to an epitope more readily than it would bind to a related,similar, homologous, or analogous epitope. Thus, an antibody that“preferentially binds” to a given epitope would more likely bind to thatepitope than to a related epitope, even though such an antibody maycross-react with the related epitope.

By way of non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds said first epitope with adissociation constant (K_(D)) that is less than the antibody's K_(D) forthe second epitope. In another non-limiting example, an antibody may beconsidered to bind a first antigen preferentially if it binds the firstepitope with an affinity that is at least one order of magnitude lessthan the antibody's K_(D) for the second epitope. In anothernon-limiting example, an antibody may be considered to bind a firstepitope preferentially if it binds the first epitope with an affinitythat is at least two orders of magnitude less than the antibody's K_(D)for the second epitope.

In another non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds the first epitope with an offrate (k(off)) that is less than the antibody's k(off) for the secondepitope. In another non-limiting example, an antibody may be consideredto bind a first epitope preferentially if it binds the first epitopewith an affinity that is at least one order of magnitude less than theantibody's k(off) for the second epitope. In another non-limitingexample, an antibody may be considered to bind a first epitopepreferentially if it binds the first epitope with an affinity that is atleast two orders of magnitude less than the antibody's k(off) for thesecond epitope.

An antibody or antigen-binding fragment, variant, or derivativedisclosed herein may be said to bind a target polypeptide disclosedherein or a fragment or variant thereof with an off rate (k(off)) ofless than or equal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹, or 10⁻³sec⁻¹. More preferably, an antibody of the invention may be said to binda target polypeptide disclosed herein or a fragment or variant thereofwith an off rate (k(off)) less than or equal to 5×10⁻⁴ sec⁻¹, 10⁻⁴sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹, 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷sec⁻¹, or 10⁻⁷ sec⁻¹.

An antibody or antigen-binding fragment, variant, or derivative thereofdisclosed herein may be said to bind a target polypeptide disclosedherein or a fragment or variant thereof with an on rate (k(on)) ofgreater than or equal to 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹,or 5×10⁴ M⁻¹ sec⁻¹. More preferably, an antibody of the invention may besaid to bind a target polypeptide disclosed herein or a fragment orvariant thereof with an on rate (k(on)) greater than or equal to 10⁵ M⁻¹sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, 5×10⁶ M⁻¹ sec⁻¹, or 10⁷ M⁻¹sec⁻¹.

An antibody is said to competitively inhibit binding of a referenceantibody to a given epitope if it preferentially binds to that epitopeto the extent that it blocks, to some degree, binding of the referenceantibody to the epitope. Competitive inhibition may be determined by anymethod known in the art, for example, competition ELISA assays. Anantibody may be said to competitively inhibit binding of the referenceantibody to a given epitope by at least 90%, at least 80%, at least 70%,at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with the CDR of animmunoglobulin molecule. See, e.g., Harlow et al. (1988) Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed.) pages27-28. As used herein, the term “avidity” refers to the overallstability of the complex between a population of immunoglobulins and anantigen, that is, the functional combining strength of an immunoglobulinmixture with the antigen. See, e.g., Harlow at pages 29-34. Avidity isrelated to both the affinity of individual immunoglobulin molecules inthe population with specific epitopes, and also the valencies of theimmunoglobulins and the antigen. For example, the interaction between abivalent monoclonal antibody and an antigen with a highly repeatingepitope structure, such as a polymer, would be one of high avidity.

Anti-CD20 antibodies or antigen-binding fragments, variants, orderivatives thereof of the invention may also be described or specifiedin terms of their cross-reactivity. As used herein, the term“cross-reactivity” refers to the ability of an antibody, specific forone antigen, to react with a second antigen; a measure of relatednessbetween two different antigenic substances. Thus, an antibody is crossreactive if it binds to an epitope other than the one that induced itsformation. The cross reactive epitope generally contains many of thesame complementary structural features as the inducing epitope, and insome cases, may actually fit better than the original.

For example, certain antibodies have some degree of cross-reactivity, inthat they bind related, but non-identical epitopes, e.g., epitopes withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be said to have littleor no cross-reactivity if it does not bind epitopes with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be deemed “highlyspecific” for a certain epitope, if it does not bind any other analog,ortholog, or homolog of that epitope.

Anti-CD20 antibodies or antigen-binding fragments, variants orderivatives thereof of the invention may also be described or specifiedin terms of their binding affinity to a polypeptide of the invention.Preferred binding affinities include those with a dissociation constantor Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M,5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M,5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M,10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

Anti-CD20 antibodies or antigen-binding fragments, variants orderivatives thereof of the invention may be “multispecific,” e.g.,bispecific, trispecific, or of greater multispecificity, meaning that itrecognizes and binds to two or more different epitopes present on one ormore different antigens (e.g., proteins) at the same time. Thus, whetheran anti-CD20 antibody is “monospecific” or “multispecific,” e.g.,“bispecific,” refers to the number of different epitopes with which abinding polypeptide reacts. Multispecific antibodies may be specific fordifferent epitopes of a target polypeptide described herein or may bespecific for a target polypeptide as well as for a heterologous epitope,such as a heterologous polypeptide or solid support material.

As used herein the term “valency” refers to the number of potentialbinding domains, e.g., antigen binding domains, present in an anti-CD20antibody, binding polypeptide, or antibody. Each binding domainspecifically binds one epitope. When an anti-CD20 antibody, bindingpolypeptide, or antibody comprises more than one binding domain, eachbinding domain may specifically bind the same epitope, for an antibodywith two binding domains, termed “bivalent monospecific,” or todifferent epitopes, for an antibody with two binding domains, termed“bivalent bispecific.” An antibody may also be bispecific and bivalentfor each specificity (termed “bispecific tetravalent antibodies”). Inanother embodiment, tetravalent minibodies or domain deleted antibodiescan be made.

Bispecific bivalent antibodies, and methods of making them, aredescribed, for instance in U.S. Pat. Nos. 5,731,168; 5,807,706;5,821,333; and U.S. Patent Appl. Publ. Nos. 2003/020734 and2002/0155537, the disclosures of all of which are incorporated byreference herein. Bispecific tetravalent antibodies, and methods ofmaking them are described, for instance, in WO 02/096948 and WO00/44788, the disclosures of both of which are incorporated by referenceherein. See generally, PCT publications WO 93/17715; WO 92/08802; WO91/00360; WO 92/05793; Tutt et al. (1991) J. Immunol. 147:60-69; U.S.Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819;Kostelny et al. (1992) J. Immunol. 148: 1547-1553.

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “V_(H) domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “C_(H)1 domain” includes the first (most amino terminal)constant region domain of an immunoglobulin heavy chain. The C_(H)1domain is adjacent to the V_(H) domain and is amino terminal to thehinge region of an immunoglobulin heavy chain molecule.

As used herein the term “C_(H)2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat E A et al. op. cit. The C_(H)2 domain is unique in that it isnot closely paired with another domain. Rather, two N-linked branchedcarbohydrate chains are interposed between the two C_(H)2 domains of anintact native IgG molecule. It is also well documented that the C_(H)3domain extends from the C_(H)2 domain to the C-terminal of the IgGmolecule and comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the C_(H)1 domain to the C_(H)2 domain. Thishinge region comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux et al. (1998) J.Immunol. 161:4083).

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the C_(H)1 and C_(L)regions are linked by a disulfide bond and the two heavy chains arelinked by two disulfide bonds at positions corresponding to 239 and 242using the Kabat numbering system (position 226 or 229, EU numberingsystem).

As used herein, the term “chimeric antibody” will be held to mean anyantibody wherein the immunoreactive region or site is obtained orderived from a first species and the constant region (which may beintact, partial or modified in accordance with the instant invention) isobtained from a second species. In preferred embodiments the targetbinding region or site will be from a non-human source (e.g., mouse orprimate) and the constant region is human.

As used herein, the term “engineered antibody” refers to an antibody inwhich the variable domain in either the heavy or light chain or both isaltered by at least partial replacement of one or more CDRs from anantibody of known specificity and, if necessary, by partial frameworkregion replacement and sequence changing. Although the CDRs may bederived from an antibody of the same class or even subclass as theantibody from which the framework regions are derived, it is envisagedthat the CDRs will be derived from an antibody of different class andpreferably from an antibody from a different species. An engineeredantibody in which one or more “donor” CDRs from a non-human antibody ofknown specificity is grafted into a human heavy or light chain frameworkregion is referred to herein as a “humanized antibody.” It may not benecessary to replace all of the CDRs with the complete CDRs from thedonor variable domain to transfer the antigen binding capacity of onevariable domain to another. Rather, it may only be necessary to transferthose residues that are necessary to maintain the activity of the targetbinding site.

It is further recognized that the framework regions within the variabledomain in a heavy or light chain, or both, of a humanized antibody maycomprise solely residues of human origin, in which case these frameworkregions of the humanized antibody are referred to as “fully humanframework regions.” Alternatively, one or more residues of the frameworkregion(s) of the donor variable domain can be engineered within thecorresponding position of the human framework region(s) of a variabledomain in a heavy or light chain, or both, of a humanized antibody ifnecessary to maintain proper binding or to enhance binding to the CD20antigen. A human framework region that has been engineered in thismanner would thus comprise a mixture of human and donor frameworkresidues, and is referred to herein as a “partially human frameworkregion.” Given the explanations set forth in, e.g., U.S. Pat. Nos.5,585,089, 5,693,761, 5,693,762, and 6,180,370, it will be well withinthe competence of those skilled in the art, either by carrying outroutine experimentation or by trial and error testing to obtain afunctional engineered or humanized antibody.

For example, humanization of an anti-CD20 antibody can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substituting rodentor mutant rodent CDRs or CDR sequences for the corresponding sequencesof a human anti-CD20 antibody. See also U.S. Pat. Nos. 5,225,539;5,585,089; 5,693,761; 5,693,762; 5,859,205; herein incorporated byreference. The resulting humanized anti-CD20 antibody would comprise atleast one rodent or mutant rodent CDR within the fully human frameworkregions of the variable domain of the heavy and/or light chain of thehumanized antibody. In some instances, residues within the frameworkregions of one or more variable domains of the humanized anti-CD20antibody are replaced by corresponding non-human (for example, rodent)residues (see, for example, U.S. Pat. Nos. 5,585,089; 5,693,761;5,693,762; and 6,180,370), in which case the resulting humanizedanti-CD20 antibody would comprise partially human framework regionswithin the variable domain of the heavy and/or light chain.

Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance (e.g., toobtain desired affinity). In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDRs correspond tothose of a non-human immunoglobulin and all or substantially all of theframework regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones et al. (1986) Nature331:522-525; Riechmann et al. (1988) Nature 332:323-329; and Presta(1992) Curr. Op. Struct. Biol. 2:593-596; herein incorporated byreference. Accordingly, such “humanized” antibodies may includeantibodies wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some frameworkresidues are substituted by residues from analogous sites in rodentantibodies. See, for example, U.S. Pat. Nos. 5,225,539; 5,585,089;5,693,761; 5,693,762; 5,859,205. See also U.S. Pat. No. 6,180,370, andInternational Publication No. WO 01/27160, where humanized antibodiesand techniques for producing humanized antibodies having improvedaffinity for a predetermined antigen are disclosed.

As used herein, the term “properly folded polypeptide” includespolypeptides (e.g., anti-CD20 antibodies) in which all of the functionaldomains comprising the polypeptide are distinctly active. As usedherein, the term “improperly folded polypeptide” includes polypeptidesin which at least one of the functional domains of the polypeptide isnot active. In one embodiment, a properly folded polypeptide comprisespolypeptide chains linked by at least one disulfide bond and,conversely, an improperly folded polypeptide comprises polypeptidechains not linked by at least one disulfide bond.

As used herein, the term “engineered” includes manipulation of nucleicacid or polypeptide molecules by synthetic means (e.g., by recombinanttechniques, in vitro peptide synthesis, by enzymatic or chemicalcoupling of peptides or some combination of these techniques).

As used herein, the terms “linked,” “fused,” or “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components, by whatever means, including chemicalconjugation or recombinant means. An “in-frame fusion” refers to thejoining of two or more polynucleotide open reading frames (ORFs) to forma continuous longer ORF, in a manner that maintains the correcttranslational reading frame of the original ORFs. Thus, a recombinantfusion protein is a single protein containing two or more segments thatcorrespond to polypeptides encoded by the original ORFs (which segmentsare not normally so joined in nature). Although the reading frame isthus made continuous throughout the fused segments, the segments may bephysically or spatially separated by, for example, in-frame linkersequence. For example, polynucleotides encoding the CDRs of animmunoglobulin variable region may be fused, in-frame, but be separatedby a polynucleotide encoding at least one immunoglobulin frameworkregion or additional CDR regions, as long as the “fused” CDRs areco-translated as part of a continuous polypeptide.

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide in an amino to carboxylterminal direction in which residues that neighbor each other in thesequence are contiguous in the primary structure of the polypeptide.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, a polypeptide. The process includesany manifestation of the functional presence of the gene within the cellincluding, without limitation, gene knockdown as well as both transientexpression and stable expression. It includes without limitationtranscription of the gene into messenger RNA (mRNA), and the translationof such mRNA into polypeptide(s). If the final desired product is abiochemical, expression includes the creation of that biochemical andany precursors. Expression of a gene produces a “gene product.” As usedherein, a gene product can be either a nucleic acid, e.g., a messengerRNA produced by transcription of a gene, or a polypeptide which istranslated from a transcript. Gene products described herein furtherinclude nucleic acids with post transcriptional modifications, e.g.,polyadenylation, or polypeptides with post translational modifications,e.g., methylation, glycosylation, the addition of lipids, associationwith other protein subunits, proteolytic cleavage, and the like.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the progression of multiplesclerosis. Beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo, sports, or pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, andso on.

As used herein, phrases such as “a subject that would benefit fromadministration of an anti-CD20 antibody” and “an animal in need oftreatment” includes subjects, such as mammalian subjects, that wouldbenefit from administration of an anti-CD20 antibody used, e.g., fordetection of an anti-CD20 polypeptide (e.g., for a diagnostic procedure)and/or for treatment, i.e., palliation or prevention of a disease, withan anti-CD20 antibody. As described in more detail herein, the anti-CD20antibody can be used in unconjugated form or can be conjugated, e.g., toa drug, prodrug, or an isotope.

II. Target Polypeptide Description

The B1 (CD20) molecule is a phosphoprotein of approximately 35,000Daltons on the surface of human B lymphocytes that may serve a centralrole in the humoral immune response by regulating B-cell proliferationand differentiation. The DNA sequence that encodes the CD20 molecule wasisolated and the amino acid sequence of CD20 has been determined. See,Tedder et al. (1988) Proc. Natl. Acad. Sci. USA 85:208-212. Synonyms ofCD20, as recognized in the art, include B-lymphocyte antigen CD20,B-lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5.

The antibodies of the invention have binding specificity to the CD20human B-cell surface antigen. The antigen is a polypeptide or comprisesa polypeptide bound by the 2H7 monoclonal antibody described in Clark etal. (1985) Proc. Natl. Acad. Sci. U.S.A. 82: 1766-1770. This antigen isa phosphoprotein designated (Bp35(CD20)) and is only expressed on cellsof the B cell lineage. Murine monoclonal antibodies to this antigen havebeen made before and are described in Clark et al., supra; see alsoStashenko et al. (1980) J. Immunol. 125:1678-1685.

III. Anti-CD20 Antibodies

In one embodiment, the present invention is directed to anti-CD20antibodies, including antigen-binding fragments, variants, orderivatives thereof. The antibodies of the invention are anti-CD20antibodies that have been optimized for enhanced activity. As usedherein, the term “anti-CD20 antibody” is an antibody that specificallyrecognizes a cell surface non-glycosylated phosphoprotein ofapproximately 35,000 Daltons, typically designated as the humanB-lymphocyte restricted differentiation antigen Bp35, commonly referredto as CD20. The antigen is expressed on greater than 90% of B-cellnon-Hodgkin's lymphomas but is not found on hematopoietic stem cells,pro-B cells, normal plasma cells, or other normal tissues. CD20regulates an early step in the activation process for cell proliferationand differentiation. More specifically, the antibodies of the inventionbind the same epitope as bound by the 2H7 monoclonal antibody describedabove.

The antibodies of the invention are optimized based on the monoclonalantibody (mAb) C2B8, a chimeric murine/human anti-CD20 antibody asdisclosed in U.S. Pat. No. 5,736,137 and (Reff et al. (1994) Blood83:435-445), herein incorporated by reference. The antibody sequences ofthe invention comprise modified CDRs that when engineered into thevariable domains of light and heavy chains of an anti-CD20 antibodyresult in enhanced CDC activity of the antibody while maintainingbinding specificity and ability to mediate apoptosis as compared torituximab.

As noted, the anti-CD20 antibodies of the invention exhibit enhanced CDCactivity as compared to rituximab. Rituximab is a chimeric murine/humananti-CD20 monoclonal antibody (IDEC-C2B8; IDEC Pharmaceutical Corp., SanDiego, Calif.; commercially available under the tradename Rituxan®)containing human IgG1 and kappa constant regions with murine variableregions isolated from a murine anti-CD20 monoclonal antibody, IDEC-2B8(Reff et al. (1994) Blood 83:435-445). Rituximab is used for treatmentof relapsed B cell low-grade or follicular non-Hodgkin's lymphoma (NHL).While not bound to any mechanism of action, anti-CD20 antibodies bind tothe CD20 antigen and mechanisms of cell lysis includecomplement-dependent cytotoxicity (CDC) and antibody-dependent cellmediated cytotoxicity (ADCC). Therefore, the discovery of anti-CD20antibodies with superior CDC and/or ADCC activity, and/or increasedbinding affinity compared to rituximab (also referred to herein asRituxan®) will potentially improve methods of cancer therapy for B celllymphomas, particularly B cell non-Hodgkin's lymphoma. The antibodiesare compared in assays in equivalent amounts. By “equivalent amount” ofthe anti-CD20 antibody of the invention and rituximab is intended thesame dose is used for each antibody.

Binding affinity of these novel anti-CD20 antibodies for CD20 isincreased by at least about 50%, about 75%, about 100%, about 125%relative to that observed for rituximab using a CDC off-rate assay. In anon-radioactive CDC assay, at least about 2 times, about 3 times, up toabout 4 times more rituximab is needed to mediate the same level of cellkilling on three different NHL lines than is needed using an anti-CD20antibody of the invention.

The anti-CD20 antibodies of the invention comprise at least oneoptimized complementarity-determining region (CDR). By “optimized CDR”is intended that the CDR has been modified and optimized sequencesselected based on the improved binding affinity and/or improved CDCactivity that is imparted to an anti-CD20 antibody comprising theoptimized CDR. The modifications involve replacement of amino acidresidues within the CDR such that an anti-CD20 antibody retainsspecificity for the CD20 antigen and has improved binding affinityand/or improved CDC activity. CDC activity of an anti-CD20 antibody ofthe invention is improved as compared to rituxamab in a functional assayas described in U.S. Application Publication No. 2004/0167319 A1, hereinincorporated by reference. The novel anti-CD20 antibodies of theinvention and suitable antigen-binding fragments, variants, andderivatives thereof also exhibit ADCC and apoptosis activity that is atleast similar to that exhibited by rituximab, as measured in standardassays, for example, those described in U.S. Application Publication No.2004/0167319 A1. The optimized CDRs of the invention are utilized inV_(H) and V_(L) domains of the heavy and light chains, respectively, ofanti-CD20 antibodies. Exemplary anti-CD20 antibodies of the inventioncomprise a V_(H) domain selected from the group consisting of SEQ IDNOS:12-18 (respectively designated H1569, H1570, H1571, H1638, H1639,H1640, H1670) and/or a V_(L) domain selected from SEQ ID NOS:10 and 11(respectively designated L373 and L419).

In particular embodiments, the anti-CD20 antibodies of the inventioncomprise optimized CDRs. That is, the anti-CD20 antibodies of theinvention comprise at least one optimized CDR amino acid sequenceselected from the group consisting of SEQ ID NOS:1-8 or amino acidsequences having at least about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about99%, or 100% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOS:1-8. That is, the optimized CDRs comprise thesequences set forth in SEQ ID NOS:1-8 and the sequences of SEQ IDNOS:1-8 having at least one, two, three, four, or five amino acidsubstitutions, depending upon the CDR involved.

Thus, in some embodiments, the anti-CD20 antibodies of the inventioncomprise a V_(H) domain having at least one optimized CDR selected fromthe group consisting of:

(a) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7;

(b) a CDR1 comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID:7,where the CDR1 comprises the phenylalanine (Phe) residue at the positioncorresponding to residue 7 of SEQ ID NO:7;

(c) a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5or SEQ ID NO:6;

(d) a CDR2 comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID:5 orSEQ ID NO:6, where the CDR2 comprises the alanine (Ala) or leucine (Leu)residue at the position corresponding to residue 8 of SEQ ID NO:5 or SEQID NO:6, respectively;

(e) a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4;

(f) a CDR3 comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:1,where the CDR3 comprises at least one residue selected from the groupconsisting of: (i) the asparagine (Asn) residue at the positioncorresponding to residue 9 of SEQ ID NO:1, and (ii) the asparagine (Asn)residue at the position corresponding to residue 12 of SEQ ID NO:1;

(g) a CDR3 comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:2,where the CDR3 comprises at least one residue selected from the groupconsisting of: (i) the alanine (Ala) residue at the positioncorresponding to residue 5 of SEQ ID NO:2, (ii) the asparagine (Asn)residue at the position corresponding to residue 9 of SEQ ID NO:2, and(iii) the asparagine (Asn) residue at the position corresponding toresidue 12 of SEQ ID NO:2;

(h) a CDR3 comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:3,where the CDR3 comprises at least one residue selected from the groupconsisting of: (i) the alanine (Ala) residue at the positioncorresponding to residue 5 of SEQ ID NO:3, (ii) the asparagine (Asn)residue at the position corresponding to residue 9 of SEQ ID NO:3, and(iii) the aspartic acid (Asp) residue at the position corresponding toresidue 12 of SEQ ID NO:3;

(i) a CDR3 comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:4,where the CDR3 comprises at least one residue selected from the groupconsisting of: (i) the alanine (Ala) residue at the positioncorresponding to residue 5 of SEQ ID NO:4, (ii) the asparagine (Asn)residue at the position corresponding to residue 9 of SEQ ID NO:4; (iii)the glycine (Gly) residue at the position corresponding to residue 11 ofSEQ ID NO:4; and (iv) the asparagine (Asn) residue at the positioncorresponding to residue 12 of SEQ ID NO:4;

(j) a CDR1 comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID:7,where the CDR1 comprises at the position corresponding to residue 7 ofSEQ ID NO:7 a residue that is a conservative amino acid substitution forphenylalanine (Phe);

(k) a CDR2 comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID:5,where the CDR2 comprises at the position corresponding to residue 8 ofSEQ ID NO:5 a residue that is a conservative amino acid substitution foralanine (Ala);

(l) a CDR2 comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID:6,where the CDR2 comprises at the position corresponding to residue 8 ofSEQ ID NO:6 a residue that is a conservative amino acid substitution forleucine (Leu);

(m) a CDR3 comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:1,where the CDR3 comprises at least one residue selected from the groupconsisting of: (i) at the position corresponding to residue 9 of SEQ IDNO:1, a residue that is a conservative amino acid substitution forasparagine (Asn), and (ii) at the position corresponding to residue 12of SEQ ID NO:1, a residue that is a conservative amino acid substitutionfor asparagine (Asn);

(n) a CDR3 comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:2,where the CDR3 comprises at least one residue selected from the groupconsisting of: (i) at the position corresponding to residue 5 of SEQ IDNO:2, a residue that is a conservative amino acid substitution foralanine (Ala), wherein the conservative amino acid substitution is notglycine, (ii) at the position corresponding to residue 9 of SEQ ID NO:2,a residue that is a conservative amino acid substitution for asparagine(Asn), and (iii) at the position corresponding to residue 12 of SEQ IDNO:2, a residue that is a conservative amino acid substitution forasparagine (Asn);

(o) a CDR3 comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:3,where the CDR3 comprises at least one residue selected from the groupconsisting of: (i) at the position corresponding to residue 5 of SEQ IDNO:3, a residue that is a conservative amino acid substitution foralanine (Ala), wherein the conservative amino acid substitution is notglycine, (ii) at the position corresponding to residue 9 of SEQ ID NO:3,a residue that is a conservative amino acid substitution for asparagine(Asn), and (iii) at the position corresponding to residue 12 of SEQ IDNO:3, a residue that is a conservative amino acid substitution foraspartic acid (Asp); and

(p) a CDR3 comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:4,where the CDR3 comprises at least one residue selected from the groupconsisting of: (i) at the position corresponding to residue 5 of SEQ IDNO:4, a residue that is a conservative amino acid substitution foralanine (Ala), wherein the conservative amino acid substitution is notglycine, (ii) at the position corresponding to residue 9 of SEQ ID NO:4,a residue that is a conservative amino acid substitution for asparagine(Asn), (iii) at the position corresponding to residue 11 of SEQ ID NO:4,a residue that is a conservative amino acid substitution for glycine(Gly), and (iv) at the position corresponding to residue 12 of SEQ IDNO:4, a residue that is a conservative amino acid substitution forasparagine (Asn).

In other embodiments, the anti-CD20 antibodies of the invention comprisea V_(L) domain having at least one CDR selected from the groupconsisting of: (a) a CDR3 comprising the amino acid sequence set forthin SEQ ID NO:8; (b) a CDR3 comprising an amino acid sequence having atleast 85% sequence identity to the amino acid sequence set forth in SEQID NO:8, where the CDR3 comprises the glutamine (Gln) residue at theposition corresponding to residue 4 of SEQ ID NO:8; and (c) a CDR3comprising an amino acid sequence having at least 85% sequence identityto the amino acid sequence set forth in SEQ ID NO:8, where the CDR3comprises at the position corresponding to residue 4 of SEQ ID NO:8 aresidue that is a conservative amino acid substitution for glutamine(Gln); and (d) a CDR2 comprising the sequence set forth in SEQ ID NO:9.In some of these embodiments, the anti-CD20 antibodies of the inventioncomprise a V_(L) domain having a CDR2 comprising the sequence set forthin SEQ ID NO:9 and a CDR3 selected from the group consisting of: (a) aCDR3 comprising the amino acid sequence set forth in SEQ ID NO:8; and(b) a CDR3 comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:8,where the CDR3 comprises the glutamine (Gln) residue at the positioncorresponding to residue 4 of SEQ ID NO:8.

In yet other embodiments, the anti-CD20 antibodies of the inventioncomprise a V_(H) domain having an optimized CDR selected from the grouprecited in items (a) through (p) supra; and a V_(L) domain having atleast one CDR selected from the group consisting of: (a) a CDR3comprising the amino acid sequence set forth in SEQ ID NO:8; (b) a CDR3comprising an amino acid sequence having at least 85% sequence identityto the amino acid sequence set forth in SEQ ID NO:8, where the CDR3comprises the glutamine (Gln) residue at the position corresponding toresidue 4 of SEQ ID NO:8; (c) a CDR3 comprising an amino acid sequencehaving at least 85% sequence identity to the amino acid sequence setforth in SEQ ID NO:8, where the CDR3 comprises at the positioncorresponding to residue 4 of SEQ ID NO:8 a residue that is aconservative amino acid substitution for glutamine (Gln); and (d) a CDR2comprising the amino acid sequence set forth in SEQ ID NO:9. In some ofthese embodiments, the anti-CD20 antibodies of the invention comprise aV_(L) domain having a CDR2 comprising the sequence set forth in SEQ IDNO:9 and a CDR3 selected from the group consisting of: (a) a CDR3comprising the amino acid sequence set forth in SEQ ID NO:8; and (b) aCDR3 comprising an amino acid sequence having at least 85% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:8, where theCDR3 comprises the glutamine (Gln) residue at the position correspondingto residue 4 of SEQ ID NO:8.

In still other embodiments, the anti-CD20 antibodies of the inventioncomprise a V_(H) domain having an optimized CDR selected from the grouprecited in items (a) through (p) supra; and a V_(L) domain having thesequence set forth in SEQ ID NO:10 or SEQ ID NO:11.

In some embodiments, the anti-CD20 antibodies comprising at least one ofthe optimized CDRs of the invention are IgG1 kappa immunoglobulins. Insuch embodiments, the IgG1 kappa immunoglobulin can comprise a humanIgG1 constant region within a heavy chain of the immunoglobulin and ahuman kappa constant region within a light chain of the immunoglobulin.In particular embodiments, the IgG1 kappa immunoglobulin comprises fullyor partially human framework regions within the variable domain of theheavy chain and within the variable domain of the light chain. In otherembodiments, the IgG1 kappa immunoglobulin comprises murine frameworkregions within the variable domain of the heavy chain and within thevariable domain of the light chain.

In further embodiments of the invention, the anti-CD20 antibodies of theinvention comprise a V_(H) domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NOS:12-18 and/or a V_(L) domainhaving an amino acid sequence selected from SEQ ID NOS:10 and 11, oramino acid sequences having at least about 80%, 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99% or 100% sequence identity to a sequence set forthin SEQ ID NOS:10-18.

In yet other embodiments of the invention, the anti-CD20 antibodies ofthe invention comprise a V_(H) domain, where the V_(H) domain isselected from the group consisting of:

(a) a V_(H) domain comprising an amino acid sequence having at least 90%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOS:12-18;

(b) a V_(H) domain comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:12,where the V_(H) domain comprises at least one residue selected from thegroup consisting of: (i) the asparagine (Asn) residue at the positioncorresponding to residue 107 of SEQ ID NO:12, and (ii) the asparagine(Asn) residue at the position corresponding to residue 110 of SEQ IDNO:12;

(c) a V_(H) domain comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:13,where the V_(H) domain comprises at least one residue selected from thegroup consisting of: (i) the alanine (Ala) residue at the positioncorresponding to residue 103 of SEQ ID NO:13, (ii) the asparagine (Asn)residue at the position corresponding to residue 107 of SEQ ID NO:13,and (iii) the asparagine (Asn) residue at the position corresponding toresidue 110 of SEQ ID NO:13;

(d) a V_(H) domain comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:14,where the V_(H) domain comprises at least one residue selected from thegroup consisting of: (i) the alanine (Ala) residue at the positioncorresponding to residue 103 of SEQ ID NO:14, (ii) the asparagine (Asn)residue at the position corresponding to residue 107 of SEQ ID NO:14,and (iii) the aspartic acid (Asp) residue at the position correspondingto residue 110 of SEQ ID NO:14;

(e) a V_(H) domain comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:15,where the V_(H) domain comprises at least one residue selected from thegroup consisting of: (i) the phenylalanine (Phe) residue at the positioncorresponding to residue 31 of SEQ ID NO:15; (ii) the alanine (Ala)residue at the position corresponding to residue 103 of SEQ ID NO:15;(iii) the asparagine (Asn) residue at the position corresponding toresidue 107 of SEQ ID NO:15, and (iv) the asparagine (Asn) residue atthe position corresponding to residue 110 of SEQ ID NO:15;

(f) a V_(H) domain comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:16,where the V_(H) domain comprises at least one residue selected from thegroup consisting of: (i) the alanine (Ala) residue at the positioncorresponding to residue 57 of SEQ ID NO:16, (ii) the alanine (Ala)residue at the position corresponding to residue 103 of SEQ ID NO:16,(iii) the asparagine (Asn) residue at the position corresponding toresidue 107 of SEQ ID NO:16, and (iv) the asparagine (Asn) residue atthe position corresponding to residue 110 of SEQ ID NO:16;

(g) a V_(H) domain comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:17,where the V_(H) domain comprises at least one residue selected from thegroup consisting of: (i) the leucine (Leu) residue at the positioncorresponding to residue 57 of SEQ ID NO:17, (ii) the alanine (Ala)residue at the position corresponding to residue 103 of SEQ ID NO:17,(iii) the asparagine (Asn) residue at the position corresponding toresidue 107 of SEQ ID NO:17, and (iv) the asparagine (Asn) residue atthe position corresponding to residue 110 of SEQ ID NO:17;

(h) a V_(H) domain comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:18,where the V_(H) domain comprises at least one residue selected from thegroup consisting of: (i) the alanine (Ala) residue at the positioncorresponding to residue 103 of SEQ ID NO:18, (ii) the asparagine (Asn)residue at the position corresponding to residue 107 of SEQ ID NO:18,(iii) the glycine (Gly) residue at the position corresponding to residue109 of SEQ ID NO:18, and (iv) the asparagine (Asn) residue at theposition corresponding to residue 110 of SEQ ID NO:18;

(i) a V_(H) domain comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:12,where the V_(H) domain comprises at least one residue selected from thegroup consisting of: (i) at the position corresponding to residue 107 ofSEQ ID NO:12, a residue that is a conservative amino acid substitutionfor asparagine (Asn), and (ii) at the position corresponding to residue110 of SEQ ID NO:12, a residue that is a conservative amino acidsubstitution for asparagine (Asn);

(j) a V_(H) domain comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:13,where the V_(H) domain comprises at least one residue selected from thegroup consisting of: (i) at the position corresponding to residue 103 ofSEQ ID NO:13, a residue that is a conservative amino acid substitutionfor alanine (Ala), wherein the conservative amino acid substitution isnot glycine, (ii) at the position corresponding to residue 107 of SEQ IDNO:13, a residue that is a conservative amino acid substitution forasparagine (Asn), and (iii) at the position corresponding to residue 110of SEQ ID NO:13, a residue that is a conservative amino acidsubstitution for asparagine (Asn);

(k) a V_(H) domain comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:14,where the V_(H) domain comprises at least one residue selected from thegroup consisting of: (i) at the position corresponding to residue 103 ofSEQ ID NO:14, a residue that is a conservative amino acid substitutionfor alanine (Ala), where the conservative amino acid substitution is notglycine, (ii) at the position corresponding to residue 107 of SEQ IDNO:14, a residue that is a conservative amino acid substitution forasparagine (Asn), and (iii) at the position corresponding to residue 110of SEQ ID NO:14, a residue that is a conservative amino acidsubstitution for aspartic acid (Asp);

(l) a V_(H) domain comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:15,where the V_(H) domain comprises at least one residue selected from thegroup consisting of: (i) at the position corresponding to residue 31 ofSEQ ID NO:15, a residue that is a conservative amino acid substitutionfor phenylalanine (Phe), (ii) at the position corresponding to residue103 of SEQ ID NO:15, a residue that is a conservative amino acidsubstitution for alanine (Ala), where the conservative amino acidsubstitution is not glycine, (iii) at the position corresponding toresidue 107 of SEQ ID NO:15, a residue that is a conservative amino acidsubstitution for asparagine (Asn), and (iv) at the positioncorresponding to residue 110 of SEQ ID NO:15, a residue that is aconservative amino acid substitution for asparagine (Asn);

(m) a V_(H) domain comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:16,where the V_(H) domain comprises at least one residue selected from thegroup consisting of: (i) at the position corresponding to residue 57 ofSEQ ID NO:16, a residue that is a conservative amino acid substitutionfor alanine (Ala), (ii) at the position corresponding to residue 103 ofSEQ ID NO:16, a residue that is a conservative amino acid substitutionfor alanine (Ala), wherein the conservative amino acid substitution isnot glycine, (iii) at the position corresponding to residue 107 of SEQID NO:16, a residue that is a conservative amino acid substitution forasparagine (Asn), and (iv) at the position corresponding to residue 110of SEQ ID NO:16, a residue that is a conservative amino acidsubstitution for asparagine (Asn);

(n) a V_(H) domain comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:17,where the V_(H) domain comprises at least one residue selected from thegroup consisting of: (i) at the position corresponding to residue 57 ofSEQ ID NO:17, a residue that is a conservative amino acid substitutionfor leucine (Leu), (ii) at the position corresponding to residue 103 ofSEQ ID NO:17, a residue that is a conservative amino acid substitutionfor alanine (Ala), where the conservative amino acid substitution is notglycine, (iii) at the position corresponding to residue 107 of SEQ IDNO:17, a residue that is a conservative amino acid substitution forasparagine (Asn), and (iv) at the position corresponding to residue 110of SEQ ID NO:17, a residue that is a conservative amino acidsubstitution for asparagine (Asn); and

(o) a V_(H) domain comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:18,where the V_(H) domain comprises at least one residue selected from thegroup consisting of: (i) at the position corresponding to residue 103 ofSEQ ID NO:18, a residue that is a conservative amino acid substitutionfor alanine (Ala), where the conservative amino acid substitution is notglycine, (ii) at the position corresponding to residue 107 of SEQ IDNO:18, a residue that is a conservative amino acid substitution forasparagine (Asn), (iii) at the position corresponding to residue 109 ofSEQ ID NO:18, a residue that is a conservative amino acid substitutionfor glycine (Gly), and (iv) at the position corresponding to residue 110of SEQ ID NO:18, a residue that is a conservative amino acidsubstitution for asparagine (Asn).

In some of these embodiments, the anti-CD20 antibodies of the inventioncomprise a V_(H) domain selected from the group recited in items (a)through (o) supra; and comprise a variable light (V_(L)) domain, wherethe V_(L) domain is selected from the group consisting of:

(a) a V_(L) domain comprising the sequence set forth in SEQ ID NO:10 orSEQ ID NO:11;

(b) a V_(L) domain comprising an amino acid sequence having at least 90%sequence identity to the sequence set forth in SEQ ID NO:10 or SEQ IDNO:11;

(c) a V_(L) domain comprising an amino acid sequence having at least 85%sequence identity to the sequence set forth in SEQ ID NO:10, where theV_(L) domain comprises the CDR2 set forth in SEQ ID NO:9;

(d) a V_(L) domain comprising an amino acid sequence having at least 85%sequence identity to the sequence set forth in SEQ ID NO:11, where theV_(L) domain comprises at least one of: (i) the CDR2 set forth in SEQ IDNO:9, and (ii) the glutamine (Gln) residue at the position correspondingto residue 91 of SEQ ID NO:11; and

(e) a V_(L) domain comprising an amino acid sequence having at least 85%sequence identity to the sequence set forth in SEQ ID NO:11, where theV_(L) domain comprises at least one of: (i) the CDR2 set forth in SEQ IDNO:9, and (ii) at the position corresponding to residue 91 of SEQ IDNO:11, a conservative amino acid substitution for glutamine (Gln).

In some of these embodiments, the anti-CD20 antibodies of the inventioncomprise a V_(H) domain comprising the amino acid sequence set forth inSEQ ID NO:12 and a V_(L) domain comprising the amino acid sequence setforth in SEQ ID NO:10. In other embodiments, the anti-CD20 antibodies ofthe invention comprise a V_(H) domain comprising the amino acid sequenceset forth in SEQ ID NO:13 and a V_(L) domain comprising the amino acidsequence set forth in SEQ ID NO:10. In certain other embodiments, theanti-CD20 antibodies of the invention comprise a V_(H) domain comprisingthe amino acid sequence set forth in SEQ ID NO:14 and a V_(L) domaincomprising the amino acid sequence set forth in SEQ ID NO:10. In stillother embodiments, the anti-CD20 antibodies of the invention comprise aV_(H) domain comprising the amino acid sequence set forth in SEQ IDNO:15 and a V_(L) domain comprising the amino acid sequence set forth inSEQ ID NO:10. In yet other embodiments, the anti-CD20 antibodies of theinvention comprise a V_(H) domain comprising the amino acid sequence setforth in SEQ ID NO:16 and a V_(L) domain comprising the amino acidsequence set forth in SEQ ID NO:10. In further embodiments, theanti-CD20 antibodies of the invention comprise a V_(H) domain comprisingthe amino acid sequence set forth in SEQ ID NO:17 and a V_(L) domaincomprising the amino acid sequence set forth in SEQ ID NO:10. In stillfurther embodiments, the anti-CD20 antibodies of the invention comprisea V_(H) domain comprising the amino acid sequence set forth in SEQ IDNO:18 and a V_(L) domain comprising the amino acid sequence set forth inSEQ ID NO:10. In certain embodiments, the anti-CD20 antibodies of theinvention comprise a V_(H) domain comprising the amino acid sequence setforth in SEQ ID NO:12 and a V_(L) domain comprising the amino acidsequence set forth in SEQ ID NO:1. In other embodiments, the anti-CD20antibodies of the invention comprise a V_(H) domain comprising the aminoacid sequence set forth in SEQ ID NO:13 and a V_(L) domain comprisingthe amino acid sequence set forth in SEQ ID NO:1. In certain otherembodiments, the anti-CD20 antibodies of the invention comprise a V_(H)domain comprising the amino acid sequence set forth in SEQ ID NO:14 anda V_(L) domain comprising the amino acid sequence set forth in SEQ IDNO:11. In still other embodiments, the anti-CD20 antibodies of theinvention comprise a V_(H) domain comprising the amino acid sequence setforth in SEQ ID NO:15 and a V_(L) domain comprising the amino acidsequence set forth in SEQ ID NO: 11. In yet other embodiments, theanti-CD20 antibodies of the invention comprise a V_(H) domain comprisingthe amino acid sequence set forth in SEQ ID NO:16 and a V_(L) domaincomprising the amino acid sequence set forth in SEQ ID NO:11. In furtherembodiments, the anti-CD20 antibodies of the invention comprise a V_(H)domain comprising the amino acid sequence set forth in SEQ ID NO:17 anda V_(L) domain comprising the amino acid sequence set forth in SEQ IDNO:11. In still further embodiments, the anti-CD20 antibodies of theinvention comprise a V_(H) domain comprising the amino acid sequence setforth in SEQ ID NO:18 and a V_(L) domain comprising the amino acidsequence set forth in SEQ ID NO:11.

In yet other embodiments, the anti-CD20 antibodies of the inventioncomprise a V_(H) domain comprising an amino acid sequence having atleast 90% sequence identity to any one of the sequences set forth in SEQID NOS:12-18. In some of these embodiments, the anti-CD20 antibodies ofthe invention further comprise a V_(L) domain comprising the amino acidsequence set forth in SEQ ID NO:10 or SEQ ID NO:11.

In other embodiments, the anti-CD20 antibodies of the invention comprisethe V_(H) domain set forth in SEQ ID NO:29 (designated H1286) or a V_(H)domain having at least about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, or 100% sequence identity to the V_(H) domain set forth inSEQ ID NO:29. In some of these embodiments, these anti-CD20 antibodiesalso comprise a V_(L) domain selected from SEQ ID NOS:10 and 11(respectively designated L373 and L419).

In yet other embodiments, the anti-CD20 antibodies of the inventioncomprise a V_(H) domain selected from the group consisting of: (a) theV_(H) domain set forth in SEQ ID NO:29, and (b) a V_(H) domain having atleast about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or100% sequence identity to the V_(H) domain set forth in SEQ ID NO:29;and a V_(L) domain having at least one CDR selected from the groupconsisting of: (a) a CDR3 comprising the amino acid sequence set forthin SEQ ID NO:8; (b) a CDR3 comprising an amino acid sequence having atleast 85% sequence identity to the amino acid sequence set forth in SEQID NO:8, where the CDR3 comprises the glutamine (Gln) residue at theposition corresponding to residue 4 of SEQ ID NO:8; and (c) a CDR3comprising an amino acid sequence having at least 85% sequence identityto the amino acid sequence set forth in SEQ ID NO:8, where the CDR3comprises at the position corresponding to residue 4 of SEQ ID NO:8 aresidue that is a conservative amino acid substitution for glutamine(Gln). Such anti-CD20 antibodies can optionally comprise a CDR2comprising the sequence set forth in SEQ ID NO:9.

In still other embodiments, the anti-CD20 antibodies of the inventioncomprise a V_(H) domain selected from the group consisting of: (a) theV_(H) domain set forth in SEQ ID NO:29, and (b) a V_(H) domain having atleast about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or100% sequence identity to the V_(H) domain set forth in SEQ ID NO:29;and a V_(L) domain having at least one CDR selected from the groupconsisting of: (a) a CDR3 comprising the amino acid sequence set forthin SEQ ID NO:8; (b) a CDR3 comprising an amino acid sequence having atleast 85% sequence identity to the amino acid sequence set forth in SEQID NO:8, where the CDR3 comprises the glutamine (Gln) residue at theposition corresponding to residue 4 of SEQ ID NO:8; (c) a CDR3comprising an amino acid sequence having at least 85% sequence identityto the amino acid sequence set forth in SEQ ID NO:8, where the CDR3comprises at the position corresponding to residue 4 of SEQ ID NO:8 aresidue that is a conservative amino acid substitution for glutamine(Gln); and (d) a CDR2 comprising the amino acid sequence set forth inSEQ ID NO:9.

In some of these embodiments, the anti-CD20 antibodies of the inventioncomprise a V_(H) domain selected from the group consisting of: (a) theV_(H) domain set forth in SEQ ID NO:29, and (b) a V_(H) domain having atleast about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or100% sequence identity to the V_(H) domain set forth in SEQ ID NO:29;and a variable light (V_(L)) domain, where the V_(L) domain is selectedfrom the group consisting of: (a) a V_(L) domain comprising the sequenceset forth in SEQ ID NO:10 or SEQ ID NO:11; (b) a V_(L) domain comprisingan amino acid sequence having at least 90% sequence identity to thesequence set forth in SEQ ID NO:10 or SEQ ID NO:11; (c) a V_(L) domaincomprising an amino acid sequence having at least 85% sequence identityto the sequence set forth in SEQ ID NO:10, where the V_(L) domaincomprises the CDR2 set forth in SEQ ID NO:9; (d) a V_(L) domaincomprising an amino acid sequence having at least 85% sequence identityto the sequence set forth in SEQ ID NO:11, where the V_(L) domaincomprises at least one of: (i) the CDR2 set forth in SEQ ID NO:9, and(ii) the glutamine (Gln) residue at the position corresponding toresidue 91 of SEQ ID NO:11; and (e) a V_(L) domain comprising an aminoacid sequence having at least 85% sequence identity to the sequence setforth in SEQ ID NO:11, where the V_(L) domain comprises at least one of:(i) the CDR2 set forth in SEQ ID NO:9, and (ii) at the positioncorresponding to residue 91 of SEQ ID NO:11, a conservative amino acidsubstitution for glutamine (Gln).

Anti-CD20 antibodies sequences are known in the art. See, for example,U.S. Pat. Nos. 5,736,137; 5,776,456; 5,843,439; 5,500,362; 5,677,180;5,693,493; 5,721,108; 5,736,137; 6,120,767; 5,843,685; 5,576,184;6,399,061; and, U.S. Patent Application Publication No. 2004/0167319;all of which are herein incorporated by reference. It is recognized thatthe modifications described here can be made to any of the anti-CD20antibodies known in the art or combinations thereof. Thus murineanti-CD20 antibodies, chimeric anti-CD20 antibodies, and humanizedanti-CD20 antibodies comprising at least one of the optimized CDRsdescribed herein are contemplated by the present invention. Anti-CD20antibodies engineered with these modifications and combinations can betested for the enhanced activity by assays known in the art anddescribed herein. Methods for measuring anti-CD20 antibody bindingspecificity include, but are not limited to, standard competitivebinding assays, assays for monitoring immunoglobulin secretion by Bcells, B cell proliferation assays, Banchereau-Like B cell proliferationassays, T cell helper assays for antibody production, co-stimulation ofB cell proliferation assays, and assays for up-regulation of B cellactivation markers. See, for example, such assays disclosed in WO00/75348 and U.S. Pat. No. 6,087,329. For CDC, ADCC, and apoptosisassays, see, for example, Subbramanian et al. (2002) J. Clin. Microbiol.40:2141-2146; Ahman et al. (1994) J. Immunol. Methods 36:243-254;Brezicka et al. (2000) Cancer Immunol. Immunother. 49:235-242;Gazzano-Santoro et al. (1997) J. Immunol. Methods 202:163-171; Prang etal. (2005) British J. Cancer 92:342-349; Shan et al. (1998) Blood92:3756-3771; Ghetie et al. (2001) Blood 97:1392-1398; and, Mathas etal. (2000) Cancer Research 60:7170-7176; all of which are hereinincorporated by reference.

Particular anti-CD20 antibodies of the invention include a V_(H) domainhaving an amino acid sequence selected from one of SEQ ID NOS:12-18 orvariants thereof paired with a V_(L) domain having an amino acidsequence of SEQ ID NO:10 or 11 or variants thereof, in any combination.Anti-CD20 antibodies of the invention include antibodies comprising aV_(H) domain having an amino acid sequence selected from the groupconsisting of SEQ ID NOS:12-18 and a V_(L) domain having an amino acidsequence selected from SEQ ID NO:10 or SEQ ID NO:11.

Suitable biologically active variants of the anti-CD20 antibodies can beused in the methods of the present invention. Such variants will retainthe desired binding properties of the parent anti-CD20 antibody. Methodsfor making antibody variants are generally available in the art.

For example, amino acid sequence variants of an anti-CD20 antibody, anantibody region, for example the CDRs (SEQ ID NOS:1-8), or an antibodyvariable domain of a heavy or light chain, for example the V_(H) domainset forth in any one of SEQ ID NOS:12-18 or the V_(L) domain set forthin SEQ ID NO:10 or SEQ ID NO:11, described herein, can be prepared bymutations in the cloned DNA sequence encoding the amino acid sequence ofinterest. Methods for mutagenesis and nucleotide sequence alterationsare well known in the art. See, for example, Walker and Gaastra, eds.(1983) Techniques in Molecular Biology (MacMillan Publishing Company,New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkelet al. (1987) Methods Enzymol. 154:367-382; Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.); U.S.Pat. No. 4,873,192; and the references cited therein; hereinincorporated by reference. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the polypeptideof interest may be found in the model of Dayhoff et al. (1978) in Atlasof Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), pp. 345-352, herein incorporated by reference in itsentirety. The model of Dayhoff et al. uses the Point Accepted Mutation(PAM) amino acid similarity matrix (PAM 250 matrix) to determinesuitable conservative amino acid substitutions. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be preferred. Examples of conservative aminoacid substitutions as taught by the PAM 250 matrix of the Dayhoff et al.model include, but are not limited to, Gly

Ala, Val

Ile

Leu, Asp

Glu, Lys

Arg, Asn

Gln, and Phe

Trp

Tyr.

In constructing variants of the anti-CD20 antibody polypeptides ofinterest, modifications are made such that variants continue to possessthe desired properties, i.e., being capable of specifically binding to ahuman CD20 antigen expressed on the surface of a human cell, and havingenhanced function, particularly increased CDC activity and/or increasedbinding affinity for the CD20 antigen, as described herein. Obviously,any mutations made in the DNA encoding the variant polypeptide must notplace the sequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure. SeeEP Patent Application Publication No. 75,444.

In addition, the constant region of an anti-CD20 antibody can be mutatedto alter effector function in a number of ways. For example, see U.S.Pat. No. 6,737,056B1 and U.S. Patent Application Publication No.2004/0132101A1, which disclose Fc mutations that optimize antibodybinding to Fc receptors.

Preferably, variants of a reference anti-CD20 antibody have amino acidsequences that have at least about 80%, about 85%, about 88%, about 90%,about 91%, about 92%, about 93%, about 94%, or about 95% sequenceidentity to the amino acid sequence for the reference anti-CD20 antibodymolecule or to a shorter portion of the reference antibody molecule.More preferably, the molecules share at least about 96%, about 97%,about 98%, or about 99% sequence identity. When discussed herein,whether any particular polypeptide, including the CDRs, V_(H) domains,and V_(L) domains disclosed herein, is at least about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or even about 100% identical to another polypeptide can be determinedusing methods and computer programs/software known in the art such as,but not limited to, the BESTFIT program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711). BESTFIT uses thelocal homology algorithm of Smith and Waterman (1981) Adv. Appl. Math.2:482-489, to find the best segment of homology between two sequences.When using BESTFIT or any other sequence alignment program to determinewhether a particular sequence is, for example, 95% identical to areference sequence according to the present invention, the parametersare set, of course, such that the percentage of identity is calculatedover the full length of the reference polypeptide sequence and that gapsin homology of up to 5% of the total number of amino acids in thereference sequence are allowed.

For purposes of the present invention, percent sequence identity isdetermined using the Smith-Waterman homology search algorithm using anaffine gap search with a gap open penalty of 12 and a gap extensionpenalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology searchalgorithm is taught in Smith and Waterman (1981) Adv. Appl. Math.2:482-489. A variant may, for example, differ from the referenceanti-CD20 antibody by as few as 1 to 15 amino acid residues, as few as 1to 10 amino acid residues, such as 6-10, as few as 5, as few as 4, 3, 2,or even 1 amino acid residue.

With respect to optimal alignment of two amino acid sequences, thecontiguous segment of the variant amino acid sequence may haveadditional amino acid residues or deleted amino acid residues withrespect to the reference amino acid sequence. The contiguous segmentused for comparison to the reference amino acid sequence will include atleast 20 contiguous amino acid residues, and may be 30, 40, 50, or moreamino acid residues. Corrections for sequence identity associated withconservative residue substitutions or gaps can be made (seeSmith-Waterman homology search algorithm).

When any two polypeptide sequences are optimally aligned for comparison,it is recognized that residues appearing opposite of one another withinthe alignment occupy positions within their respective polypeptides thatcorrespond to one another. Such positions are referred to herein as“corresponding positions” and the residues residing at correspondingpositions are referred to as “corresponding residues” or residues that“correspond” to one another. Thus, for example, where a polypeptide ofinterest is optimally aligned to a reference polypeptide sequencehaving, for example, 10 residues, the residue within the polypeptide ofinterest appearing opposite residue 5 of the reference sequence isreferred to as the “residue at the position corresponding to residue 5”of the reference sequence.

The precise chemical structure of a polypeptide capable of specificallybinding CD20 and retaining the desired increase in CDC activity and/orincreased binding affinity for the CD20 antigen depends on a number offactors. As ionizable amino and carboxyl groups are present in themolecule, a particular polypeptide may be obtained as an acidic or basicsalt, or in neutral form. All such preparations that retain theirbiological activity when placed in suitable environmental conditions areincluded in the definition of anti-CD20 antibodies as used herein.Further, the primary amino acid sequence of the polypeptide may beaugmented by derivatization using sugar moieties (glycosylation) or byother supplementary molecules such as lipids, phosphate, acetyl groupsand the like. It may also be augmented by conjugation with saccharides.Certain aspects of such augmentation are accomplished throughpost-translational processing systems of the producing host; other suchmodifications may be introduced in vitro. In any event, suchmodifications are included in the definition of an anti-CD20 antibodyused herein so long as the desired properties of the anti-CD20 antibodyare not destroyed. It is expected that such modifications mayquantitatively or qualitatively affect the activity, either by enhancingor diminishing the activity of the polypeptide, in the various assays.Further, individual amino acid residues in the chain may be modified byoxidation, reduction, or other derivatization, and the polypeptide maybe cleaved to obtain fragments that retain activity. Such alterationsthat do not destroy the desired properties (i.e., binding specificityfor CD20, and increased CDC activity and/or increased binding affinityfor the CD20 antigen) do not remove the polypeptide sequence from thedefinition of anti-CD20 antibodies of interest as used herein.

The art provides substantial guidance regarding the preparation and useof polypeptide variants. In preparing the anti-CD20 antibody variants,one of skill in the art can readily determine which modifications to thenative protein nucleotide or amino acid sequence will result in avariant that is suitable for use as a therapeutically active componentof a pharmaceutical composition used in the methods of the presentinvention.

In certain anti-CD20 antibodies, the Fc portion may be mutated todecrease effector function using techniques known in the art. Forexample, the deletion or inactivation (through point mutations or othermeans) of a constant region domain may reduce Fc receptor binding of thecirculating modified antibody thereby increasing tumor localization. Inother cases it may be that constant region modifications consistent withthe instant invention moderate complement binding and thus reduce theserum half life and nonspecific association of a conjugated cytotoxin.Yet other modifications of the constant region may be used to modifydisulfide linkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or antibodyflexibility. The resulting physiological profile, bioavailability andother biochemical effects of the modifications, such as tumorlocalization, biodistribution and serum half-life, may easily bemeasured and quantified using well known immunological techniqueswithout undue experimentation.

Anti-CD20 antibodies of the invention also include derivatives that aremodified, e.g., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodyfrom specifically binding to its cognate epitope. For example, but notby way of limitation, the antibody derivatives include antibodies thathave been modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a side chain witha similar charge. Families of amino acid residues having side chainswith similar charges have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity (e.g., theability to bind an anti-CD20 polypeptide).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations may be silent or neutral missense mutations, i.e., have no, orlittle, effect on an antibody's ability to bind antigen. These types ofmutations may be useful to optimize codon usage, or improve ahybridoma's antibody production. Alternatively, non-neutral missensemutations may alter an antibody's ability to bind antigen. The locationof most silent and neutral missense mutations is likely to be in theframework regions, while the location of most non-neutral missensemutations is likely to be in CDR, though this is not an absoluterequirement. One of skill in the art would be able to design and testmutant molecules with desired properties such as no alteration inantigen binding activity or alteration in binding activity (e.g.,improvements in antigen binding activity or change in antibodyspecificity). Following mutagenesis, the encoded protein may routinelybe expressed and the functional and/or biological activity of theencoded protein, (e.g., ability to immunospecifically bind at least oneepitope of a CD20 polypeptide) can be determined using techniquesdescribed herein or by routinely modifying techniques known in the art.

IV. Polynucleotides Encoding Anti-CD20 Antibodies

The present invention also provides for nucleic acid molecules encodinganti-CD20 antibodies of the invention, or antigen-binding fragments,variants, or derivatives thereof.

In one embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable domain(V_(H) domain), where at least one of the CDRs of the V_(H) domain hasan amino acid sequence that is at least about 80%, about 85%, about 90%,about 95%, about 96%, about 97%, about 98%, about 99%, or identical toany one of SEQ ID NOS:1-7.

In other embodiments, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin V_(H) domain, where at least oneof the CDRs of the V_(H) domain is selected from the group consistingof: (a) a CDR1 comprising the amino acid sequence set forth in SEQ IDNO:7; (b) a CDR1 comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID:7,where the CDR1 comprises the phenylalanine (Phe) residue at the positioncorresponding to residue 7 of SEQ ID NO:7; (c) a CDR2 comprising theamino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6; (d) a CDR2comprising an amino acid sequence having at least 85% sequence identityto the amino acid sequence set forth in SEQ ID:5 or SEQ ID NO:6, wherethe CDR2 comprises the alanine (Ala) or leucine (Leu) residue at theposition corresponding to residue 8 of SEQ ID NO:5 or SEQ ID NO:6,respectively; (e) a CDR3 comprising the amino acid sequence set forth inSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; (f) a CDR3comprising an amino acid sequence having at least 85% sequence identityto the amino acid sequence set forth in SEQ ID NO:1, where the CDR3comprises at least one residue selected from the group consisting of:(i) the asparagine (Asn) residue at the position corresponding toresidue 9 of SEQ ID NO:1, and (ii) the asparagine (Asn) residue at theposition corresponding to residue 12 of SEQ ID NO:1; (g) a CDR3comprising an amino acid sequence having at least 85% sequence identityto the amino acid sequence set forth in SEQ ID NO:2, where the CDR3comprises at least one residue selected from the group consisting of (i)the alanine (Ala) residue at the position corresponding to residue 5 ofSEQ ID NO:2, (ii) the asparagine (Asn) residue at the positioncorresponding to residue 9 of SEQ ID NO:2, and (iii) the asparagine(Asn) residue at the position corresponding to residue 12 of SEQ IDNO:2; (h) a CDR3 comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:3,where the CDR3 comprises at least one residue selected from the groupconsisting of: (i) the alanine (Ala) residue at the positioncorresponding to residue 5 of SEQ ID NO:3, (ii) the asparagine (Asn)residue at the position corresponding to residue 9 of SEQ ID NO:3, and(iii) the aspartic acid (Asp) residue at the position corresponding toresidue 12 of SEQ ID NO:3; and (i) a CDR3 comprising an amino acidsequence having at least 85% sequence identity to the amino acidsequence set forth in SEQ ID NO:4, where the CDR3 comprises at least oneresidue selected from the group consisting of: (i) the alanine (Ala)residue at the position corresponding to residue 5 of SEQ ID NO:4, (ii)the asparagine (Asn) residue at the position corresponding to residue 9of SEQ ID NO:4, (iii) the glycine (Gly) residue at the positioncorresponding to residue 11 of SEQ ID NO:4, and (iv) the asparagine(Asn) residue at the position corresponding to residue 12 of SEQ IDNO:4; wherein an anti-CD20 antibody comprising the encoded V_(H) domainspecifically or preferentially binds to CD20.

In some embodiments, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin V_(H) domain, where at least oneof the CDRs of the V_(H) domain is selected from the group consistingof: (a) a CDR1 comprising the sequence set forth in SEQ ID NO:7; (b) aCDR2 comprising the sequence set forth in SEQ ID NO:5 or SEQ ID NO:6;and (c) a CDR3 comprising the sequence set forth in SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, or SEQ ID NO:4. In particular embodiments, theisolated polynucleotide encoding an immunoglobulin V_(H) domaincomprises at least one of the CDR-encoding sequences selected from thegroup consisting of SEQ ID NO:24 (encoding optimized CDR3 of SEQ IDNO:1), SEQ ID NO:25 (encoding optimized CDR3 of SEQ ID NO:2), SEQ IDNO:26 (encoding optimized CDR3 of SEQ ID NO:3), and SEQ ID NO:27(encoding optimized CDR2 of SEQ ID NO:5).

In a further embodiment, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a V_(H) domain that has an amino acid sequencethat is at least about 80%, about 85%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about99%, or 100% identical to a reference V_(H) domain polypeptide sequenceselected from the group consisting of SEQ ID NOS:12-18 and 29, whereinan anti-CD20 antibody comprising the encoded V_(H) domain specificallyor preferentially binds to CD20.

In yet other embodiments, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a V_(H) domain selected from the group consistingof: (a) a V_(H) domain comprising an amino acid sequence having at least90% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOS:12-18 and 29; (b) a V_(H) domain comprising anamino acid sequence having at least 85% sequence identity to the aminoacid sequence set forth in SEQ ID NO:12, where the V_(H) domaincomprises at least one residue selected from the group consisting of:(i) the asparagine (Asn) residue at the position corresponding toresidue 107 of SEQ ID NO:12, and (ii) the asparagine (Asn) residue atthe position corresponding to residue 110 of SEQ ID NO:12; (c) a V_(H)domain comprising an amino acid sequence having at least 85% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:13, where theV_(H) domain comprises at least one residue selected from the groupconsisting of: (i) the alanine (Ala) residue at the positioncorresponding to residue 103 of SEQ ID NO:13, (ii) the asparagine (Asn)residue at the position corresponding to residue 107 of SEQ ID NO:13,and (iii) the asparagine (Asn) residue at the position corresponding toresidue 110 of SEQ ID NO:13; (d) a V_(H) domain comprising an amino acidsequence having at least 85% sequence identity to the amino acidsequence set forth in SEQ ID NO:14, where the V_(H) domain comprises atleast one residue selected from the group consisting of: (i) the alanine(Ala) residue at the position corresponding to residue 103 of SEQ IDNO:14, (ii) the asparagine (Asn) residue at the position correspondingto residue 107 of SEQ ID NO:14, and (iii) the aspartic acid (Asp)residue at the position corresponding to residue 110 of SEQ ID NO:14;(e) a V_(H) domain comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:15,where the V_(H) domain comprises at least one residue selected from thegroup consisting of: (i) the phenylalanine (Phe) residue at the positioncorresponding to residue 31 of SEQ ID NO:15; (ii) the alanine (Ala)residue at the position corresponding to residue 103 of SEQ ID NO:15;(iii) the asparagine (Asn) residue at the position corresponding toresidue 107 of SEQ ID NO:15, and (iv) the asparagine (Asn) residue atthe position corresponding to residue 110 of SEQ ID NO:15; (f) a V_(H)domain comprising an amino acid sequence having at least 85% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:16, where theV_(H) domain comprises at least one residue selected from the groupconsisting of: (i) the alanine (Ala) residue at the positioncorresponding to residue 57 of SEQ ID NO:16, (ii) the alanine (Ala)residue at the position corresponding to residue 103 of SEQ ID NO:16,(iii) the asparagine (Asn) residue at the position corresponding toresidue 107 of SEQ ID NO:16, and (iv) the asparagine (Asn) residue atthe position corresponding to residue 110 of SEQ ID NO:16; (g) a V_(H)domain comprising an amino acid sequence having at least 85% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:17, where theV_(H) domain comprises at least one residue selected from the groupconsisting of: (i) the leucine (Leu) residue at the positioncorresponding to residue 57 of SEQ ID NO:17, (ii) the alanine (Ala)residue at the position corresponding to residue 103 of SEQ ID NO:17,(iii) the asparagine (Asn) residue at the position corresponding toresidue 107 of SEQ ID NO:17, and (iv) the asparagine (Asn) residue atthe position corresponding to residue 110 of SEQ ID NO:17; and (h) aV_(H) domain comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:18,where the V_(H) domain comprises at least one residue selected from thegroup consisting of: (i) the alanine (Ala) residue at the positioncorresponding to residue 103 of SEQ ID NO:18, (ii) the asparagine (Asn)residue at the position corresponding to residue 107 of SEQ ID NO:18,(iii) the glycine (Gly) residue at the position corresponding to residue109 of SEQ ID NO:18, and (iv) the asparagine (Asn) residue at theposition corresponding to residue 110 of SEQ ID NO:18.

In a further embodiment, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a V_(H) domain, where the nucleic acid has asequence that has at least about 80%, about 85%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99%, or 100% sequence identity to a nucleotide sequenceselected from the group consisting of SEQ ID NOS:19-22 and wherein ananti-CD20 antibody comprising the encoded V_(H) domain specifically orpreferentially binds to anti-CD20.

In other embodiments, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin light chain variable domain(V_(L) domain), where the V_(L) domain comprises a CDR with an aminoacid sequence that is at least about 80%, about 85%, about 90%, about95%, about 96%, about 97%, about 98%, about 99% or 100% identical to theCDR sequence set forth in SEQ ID NO:8.

In further embodiments, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a V_(L) domain of an immunoglobulin light chain,where the V_(L) domain comprises at least one CDR selected from thegroup consisting of: (a) a CDR3 comprising the amino acid sequence setforth in SEQ ID NO:8; (b) a CDR3 comprising an amino acid sequencehaving at least 85% sequence identity to the amino acid sequence setforth in SEQ ID NO:8, where the CDR3 comprises the glutamine (Gln)residue at the position corresponding to residue 4 of SEQ ID NO:8 and(c) a CDR2 having the amino acid sequence set forth in SEQ ID NO:9. Insome of these embodiments, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a V_(L) domain of an immunoglobulin light chain,where the V_(L) domain comprises a CDR2 having the amino acid sequenceset forth in SEQ ID NO:9 and a CDR3 selected from the group consistingof: (a) a CDR3 comprising the amino acid sequence set forth in SEQ IDNO:8; and (b) a CDR3 comprising an amino acid sequence having at least85% sequence identity to the amino acid sequence set forth in SEQ IDNO:8, where the CDR3 comprises the glutamine (Gln) residue at theposition corresponding to residue 4 of SEQ ID NO:8.

In yet a further embodiment, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a V_(L) domain that has an amino acid sequencethat is at least about 80%, about 85%, about 90%, about 95%, about 96%,about 97%, about 98%, about 99%, or 100% identical to a reference V_(L)domain polypeptide sequence set forth in SEQ ID NO:11, wherein ananti-CD20 antibody comprising the encoded V_(L) domain specifically orpreferentially binds to CD20.

In still other embodiments, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a V_(L) domain selected from the group consistingof: (a) a V_(L) domain comprising the sequence set forth in SEQ ID NO:10or SEQ ID NO:11; (b) a V_(L) domain comprising an amino acid sequencehaving at least 90% sequence identity to the sequence set forth in SEQID NO:10 or SEQ ID NO:11; (c) a V_(L) domain comprising an amino acidsequence having at least 85% sequence identity to the sequence set forthin SEQ ID NO:10, where the V_(L) domain comprises the CDR2 set forth inSEQ ID NO:9; and (d) a V_(L) domain comprising an amino acid sequencehaving at least 85% sequence identity to the sequence set forth in SEQID NO:11, where the V_(L) domain comprises at least one of: (i) the CDR2set forth in SEQ ID NO:9, and (ii) the glutamine (Gln) residue at theposition corresponding to residue 91 of SEQ ID NO:11.

Any of the polynucleotides described above may further includeadditional nucleic acids, encoding, e.g., a signal peptide to directsecretion of the encoded polypeptide, antibody constant regions asdescribed herein, or other heterologous polypeptides as describedherein. Also, as described in more detail elsewhere herein, the presentinvention includes compositions comprising the polynucleotidescomprising one or more of the polynucleotides described above. In oneembodiment, the invention includes compositions comprising a firstpolynucleotide and second polynucleotide wherein said firstpolynucleotide encodes a V_(H) domain as described herein and whereinsaid second polynucleotide encodes a V_(L) domain as described herein.Specifically a composition which comprises, consists essentially of, orconsists of a V_(H) domain-encoding polynucleotide, as set forth in anyone of SEQ ID NOS:19-22, and a V_(L) domain-encoding polynucleotide, forexample, a polynucleotide encoding the V_(L) domain as set forth in SEQID NO:10 or SEQ ID NO:11.

The present invention also includes fragments of the polynucleotides ofthe invention, as described elsewhere. Additionally polynucleotides thatencode fusion polypolypeptides, Fab fragments, and other derivatives, asdescribed herein, are also contemplated by the invention.

The polynucleotides may be produced or manufactured by any method knownin the art. For example, if the nucleotide sequence of the antibody isknown, a polynucleotide encoding the antibody may be assembled fromchemically synthesized oligonucleotides (e.g., as described in Kutmeieret al. (1994) Bio Techniques 17:242), which, briefly, involves thesynthesis of overlapping oligonucleotides containing portions of thesequence encoding the antibody, annealing and ligating of thoseoligonucleotides, and then amplification of the ligated oligonucleotidesby PCR.

Alternatively, a polynucleotide encoding an anti-CD20 antibody, orantigen-binding fragment, variant, or derivative thereof, may begenerated from nucleic acid from a suitable source. If a clonecontaining a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the antibody may be chemically synthesized or obtainedfrom a suitable source (e.g., an antibody cDNA library, or a cDNAlibrary generated from, or nucleic acid, preferably poly A+RNA, isolatedfrom, any tissue or cells expressing the antibody or other anti-CD20antibody, such as hybridoma cells selected to express an antibody) byPCR amplification using synthetic primers hybridizable to the 3′ and 5′ends of the sequence or by cloning using an oligonucleotide probespecific for the particular gene sequence to identify, e.g., a cDNAclone from a cDNA library that encodes the antibody or other anti-CD20antibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe anti-CD20 antibody, or antigen-binding fragment, variant, orderivative thereof is determined, its nucleotide sequence may bemanipulated using methods well known in the art for the manipulation ofnucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al. (1990) Molecular Cloning, A Laboratory Manual (2nd ed.;Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and Ausubel etal., eds. (1998) Current Protocols in Molecular Biology (John Wiley &Sons, NY), which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

A polynucleotide encoding an anti-CD20 antibody, or antigen-bindingfragment, variant, or derivative thereof, can be composed of anypolyribonucleotide or polydeoxyribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. For example, a polynucleotideencoding anti-CD20 antibody, or antigen-binding fragment, variant, orderivative thereof can be composed of single- and double-stranded DNA,DNA that is a mixture of single- and double-stranded regions, single-and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, a polynucleotideencoding an anti-CD20 antibody, or antigen-binding fragment, variant, orderivative thereof can be composed of triple-stranded regions comprisingRNA or DNA or both RNA and DNA. A polynucleotide encoding an anti-CD20antibody, or antigen-binding fragment, variant, or derivative thereof,may also contain one or more modified bases or DNA or RNA backbonesmodified for stability or for other reasons. “Modified” bases include,for example, tritylated bases and unusual bases such as inosine. Avariety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

An isolated polynucleotide encoding a non-natural variant of apolypeptide derived from an immunoglobulin (e.g., an immunoglobulinheavy chain portion or light chain portion) can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of the immunoglobulin such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore non-essential amino acid residues.

V. Fusion Proteins and Antibody Conjugates

As discussed in more detail elsewhere herein, anti-CD20 antibodies ofthe invention, or antigen-binding fragments, variants, or derivativesthereof, may further be recombinantly fused to a heterologouspolypeptide at the N- or C-terminus or chemically conjugated (includingcovalent and non-covalent conjugations) to polypeptides or othercompositions. For example, anti-CD20 antibodies may be recombinantlyfused or conjugated to molecules useful as labels in detection assaysand effector molecules such as heterologous polypeptides, drugs,radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.

Anti-CD20 antibodies of the invention, or antigen-binding fragments,variants, or derivatives thereof, include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody bindingCD20. For example, but not by way of limitation, the antibodyderivatives include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Additionally, thederivative may contain one or more non-classical amino acids.

Anti-CD20 antibodies of the invention, or antigen-binding fragments,variants, or derivatives thereof, can be composed of amino acids joinedto each other by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain amino acids other than the 20 gene-encodedamino acids. Anti-CD20 antibodies may be modified by natural processes,such as posttranslational processing, or by chemical modificationtechniques that are well known in the art. Such modifications are welldescribed in basic texts and in more detailed monographs, as well as ina voluminous research literature. Modifications can occur anywhere inthe anti-CD20 antibody, including the peptide backbone, the amino acidside-chains and the amino or carboxyl termini, or on moieties such ascarbohydrates. It will be appreciated that the same type of modificationmay be present in the same or varying degrees at several sites in agiven anti-CD20 antibody. Also, a given anti-CD20 antibody may containmany types of modifications. Anti-CD20 antibodies may be branched, forexample, as a result of ubiquitination, and they may be cyclic, with orwithout branching. Cyclic, branched, and branched cyclic anti-CD20antibodies may result from posttranslation natural processes or may bemade by synthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.(See, for instance, Proteins—Structure and Molecular Properties, T. E.Creighton, W. H. Freeman and Company, NY; 2nd ed. (1993); Johnson, ed.(1983) Posttranslational Covalent Modification of Proteins (AcademicPress, NY), pgs. 1-12; Seifter et al. (1990) Meth. Enzymol. 182:626-646;Rattan et al. (1992) Ann. NY Acad. Sci. 663:48-62).

The present invention also provides for fusion proteins comprising ananti-CD20 antibody, or antigen-binding fragment, variant, or derivativethereof, and a heterologous polypeptide. The heterologous polypeptide towhich the antibody is fused may be useful for function or is useful totarget the anti-CD20 polypeptide expressing cells. In one embodiment, afusion protein of the invention comprises, consists essentially of, orconsists of, a polypeptide having the amino acid sequence of any one ormore of the V_(H) domains of an antibody of the invention or the aminoacid sequence of any one or more of the V_(L) domains of an antibody ofthe invention or fragments or variants thereof, and a heterologouspolypeptide sequence. In another embodiment, a fusion protein for use inthe diagnostic and treatment methods disclosed herein comprises,consists essentially of, or consists of a polypeptide having the aminoacid sequence of any one, two, three of the CDRs of the V_(H) domain ofan anti-CD20 antibody, or fragments, variants, or derivatives thereof,or the amino acid sequence of any one, two, three of the CDRs of theV_(L) domain of an anti-CD20 antibody, or fragments, variants, orderivatives thereof, and a heterologous polypeptide sequence. In oneembodiment, the fusion protein comprises a polypeptide having the aminoacid sequence of a CDR3 of the V_(H) domain of an anti-CD20-specificantibody of the present invention, or fragment, derivative, or variantthereof, and a heterologous polypeptide sequence, which fusion proteinspecifically binds to at least one epitope of CD20. In anotherembodiment, a fusion protein comprises a polypeptide having the aminoacid sequence of at least one V_(H) domain of an anti-CD20 antibody ofthe invention and the amino acid sequence of at least one V_(L) domainof an anti-CD20 antibody of the invention or fragments, derivatives orvariants thereof, and a heterologous polypeptide sequence. Preferably,the V_(H) and V_(L) domains of the fusion protein correspond to a singlesource antibody (or scFv or Fab fragment) that specifically binds atleast one epitope of CD20. In yet another embodiment, a fusion proteinfor use in the diagnostic and treatment methods disclosed hereincomprises a polypeptide having the amino acid sequence of any one, two,three or more of the CDRs of the V_(H) domain of an anti-CD20 antibodyand the amino acid sequence of any one, two, three or more of the CDRsof the V_(L) domain of an anti-CD20 antibody, or fragments or variantsthereof, and a heterologous polypeptide sequence. Preferably, two,three, four, five, six, or more of the CDR(s) of the V_(H) domain orV_(L) domain correspond to single source antibody (or scFv or Fabfragment) of the invention. Nucleic acid molecules encoding these fusionproteins are also encompassed by the invention.

Exemplary fusion proteins reported in the literature include fusions ofthe T cell receptor (Gascoigne et al. (1987) Proc. Natl. Acad. Sci. USA84:2936-2940); CD4 (Capon et al. (1989) Nature 337:525-531; Trauneckeret al. (1989) Nature 339:68-70; Zettmeissl et al. (1990) DNA Cell Biol.USA 9:347-353; and Byrn et al. (1990) Nature 344:667-670); L-selectin(homing receptor) (Watson et al. (1990) J. Cell. Biol. 110:2221-2229;and Watson et al. (1991) Nature 349:164-167); CD44 (Aruffo et al. (1990)Cell 61:1303-1313); CD28 and B7 (Linsley et al. (1991) J. Exp. Med.173:721-730); CTLA-4 (Lisley et al. (1991) J. Exp. Med. 174:561-569);CD22 (Stamenkovic et al. (1991) Cell 66:1133-1144); TNF receptor(Ashkenazi et al. (1991) Proc. Natl. Acad. Sci. USA 88:10535-10539;Lesslauer et al. (1991) Eur. J. Immunol. 27:2883-2886; and Peppel et al.(1991) J. Exp. Med. 174:1483-1489); and IgE receptor a (Ridgway andGorman (1991) J. Cell. Biol. Vol. 115, Abstract No. 1448).

As discussed elsewhere herein, anti-CD20 antibodies of the invention, orantigen-binding fragments, variants, or derivatives thereof, may befused to heterologous polypeptides to increase the in vivo half life ofthe polypeptides or for use in immunoassays using methods known in theart. For example, in one embodiment, PEG can be conjugated to theanti-CD20 antibodies of the invention to increase their half-life invivo. See Leong et al. (2001) Cytokine 16:106; Adv. in Drug Deliv. Rev.(2002) 54:531; or Weir et al. (2002) Biochem. Soc. Transactions 30:512.

Moreover, anti-CD20 antibodies of the invention, or antigen-bindingfragments, variants, or derivatives thereof, can be fused to markersequences, such as a peptide to facilitate their purification ordetection. In preferred embodiments, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al. (1989) Proc. Natl. Acad. Sci. USA 86:821-824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al. (1984) Cell 37:767)and the “flag” tag.

Fusion proteins can be prepared using methods that are well known in theart (see for example U.S. Pat. Nos. 5,116,964 and 5,225,538). Theprecise site at which the fusion is made may be selected empirically tooptimize the secretion or binding characteristics of the fusion protein.DNA encoding the fusion protein is then transfected into a host cell forexpression.

Anti-CD20 antibodies of the present invention, or antigen-bindingfragments, variants, or derivatives thereof, may be used innon-conjugated form or may be conjugated to at least one of a variety ofmolecules, e.g., to improve the therapeutic properties of the molecule,to facilitate target detection, or for imaging or therapy of thepatient. Anti-CD20 antibodies of the invention, or antigen-bindingfragments, variants, or derivatives thereof, can be labeled orconjugated either before or after purification, when purification isperformed.

In particular, anti-CD20 antibodies of the invention, or antigen-bindingfragments, variants, or derivatives thereof, may be conjugated totherapeutic agents, prodrugs, peptides, proteins, enzymes, viruses,lipids, biological response modifiers, pharmaceutical agents, or PEG.

Those skilled in the art will appreciate that conjugates may also beassembled using a variety of techniques depending on the selected agentto be conjugated. For example, conjugates with biotin are prepared,e.g., by reacting a binding polypeptide with an activated ester ofbiotin such as the biotin N-hydroxysuccinimide ester. Similarly,conjugates with a fluorescent marker may be prepared in the presence ofa coupling agent, e.g. those listed herein, or by reaction with anisothiocyanate, preferably fluorescein-isothiocyanate. Conjugates of theanti-CD20 antibodies of the invention, or antigen-binding fragments,variants, or derivatives thereof, are prepared in an analogous manner.

The present invention further encompasses anti-CD20 antibodies of theinvention, or antigen-binding fragments, variants, or derivativesthereof, conjugated to a diagnostic or therapeutic agent. The anti-CD20antibodies, including antigen-binding fragments, variants, andderivatives thereof, can be used diagnostically to, for example, monitorthe development or progression of a disease as part of a clinicaltesting procedure to, e.g., determine the efficacy of a given treatmentand/or prevention regimen. Detection can be facilitated by coupling theanti-CD20 antibody, or antigen-binding fragment, variant, or derivativethereof, to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, radioactive materials,positron emitting metals using various positron emission tomographies,and nonradioactive paramagnetic metal ions. See, for example, U.S. Pat.No. 4,741,900 for metal ions which can be conjugated to antibodies foruse as diagnostics according to the present invention. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ¹¹¹In,⁹⁰Y, or ⁹⁹Tc.

An anti-CD20 antibody, or antigen-binding fragment, variant, orderivative thereof, may be conjugated to a therapeutic moiety such as acytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxinor cytotoxic agent includes any agent that is detrimental to cells.Examples include selenium, taxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). The conjugates of the invention canbe used for modifying a given biological response. The drug moiety isnot to be construed as limited to classical chemical therapeutic agents.For example, the drug moiety may be a protein or polypeptide possessinga desired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, alpha-interferon,beta-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

An anti-CD20 antibody, or antigen-binding fragment, variant, orderivative thereof, also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedanti-CD20 antibody is then determined by detecting the presence ofluminescence that arises during the course of a chemical reaction.Examples of particularly useful chemiluminescent labeling compounds areluminol, isoluminol, theromatic acridinium ester, imidazole, acridiniumsalt and oxalate ester.

One of the ways in which an anti-CD20 antibody, or antigen-bindingfragment, variant, or derivative thereof, can be detectably labeled isby linking the same to an enzyme and using the linked product in anenzyme immunoassay (EIA) (Voller, A., “The Enzyme Linked ImmunosorbentAssay (ELISA)” Microbiological Associates Quarterly Publication,Walkersville, Md.; Diagnostic Horizons (1978) 2:1-7; Voller et al.(1978) J. Clin. Pathol. 31:507-520; Butler (1981) Meth. Enzymol.73:482-523; Maggio, ed. (1980) Enzyme Immunoassay, CRC Press, BocaRaton, Fla.; Ishikawa et al., eds. (1981) Enzyme Immunoassay (KgakuShoin, Tokyo). The enzyme, which is bound to the anti-CD20 antibody willreact with an appropriate substrate, preferably a chromogenic substrate,in such a manner as to produce a chemical moiety which can be detected,for example, by spectrophotometric, fluorimetric or by visual means.Enzymes which can be used to detectably label the antibody include, butare not limited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Additionally, the detection can be accomplished bycolorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the anti-CD20antibody, or antigen-binding fragment, variant, or derivative thereof,it is possible to detect the antibody through the use of aradioimmunoassay (RIA) (see, for example, Weintraub (March, 1986)Principles of Radioimmunoassays, Seventh Training Course on RadioligandAssay Techniques (The Endocrine Society), which is incorporated byreference herein). The radioactive isotope can be detected by meansincluding, but not limited to, a gamma counter, a scintillation counter,or autoradiography.

An anti-CD20 antibody, or antigen-binding fragment, variant, orderivative thereof, can also be detectably labeled using fluorescenceemitting metals such as 152Eu, or others of the lanthanide series. Thesemetals can be attached to the antibody using such metal chelating groupsas diethylenetriaminepentacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

Techniques for conjugating various moieties to an anti-CD20 antibody, orantigen-binding fragment, variant, or derivative thereof, are wellknown, see, e.g., Arnon et al. (1985) “Monoclonal Antibodies forImmunotargeting of Drugs in Cancer Therapy,” in Monoclonal Antibodiesand Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.), pp.243-56; Hellstrom et al. (1987) “Antibodies for Drug Delivery,” inControlled Drug Delivery, ed. Robinson et al. (2nd ed.; Marcel Dekker,Inc.), pp. 623-53); Thorpe (1985) “Antibody Carriers of Cytotoxic Agentsin Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biologicaland Clinical Applications, ed. Pinchera et al., pp. 475-506; “Analysis,Results, and Future Prospective of the Therapeutic Use of RadiolabeledAntibody in Cancer Therapy,” in Monoclonal Antibodies for CancerDetection and Therapy, ed. Baldwin et al., Academic Press, pp. 303-16(1985); and Thorpe et al. (1982) “The Preparation and CytotoxicProperties of Antibody-Toxin Conjugates,” Immunol. Rev. 62:119-58.

VI. Expression of Antibody Polypeptides

DNA sequences that encode the light and the heavy chains of the antibodymay be made, either simultaneously or separately, using reversetranscriptase and DNA polymerase in accordance with well known methods.PCR may be initiated by consensus constant region primers or by morespecific primers based on the published heavy and light chain DNA andamino acid sequences. As discussed above, PCR also may be used toisolate DNA clones encoding the antibody light and heavy chains. In thiscase the libraries may be screened by consensus primers or largerhomologous probes, such as mouse constant region probes.

DNA, typically plasmid DNA, may be isolated from the cells usingtechniques known in the art, restriction mapped and sequenced inaccordance with standard, well known techniques set forth in detail,e.g., in the foregoing references relating to recombinant DNAtechniques. Of course, the DNA may be synthetic according to the presentinvention at any point during the isolation process or subsequentanalysis.

Following manipulation of the isolated genetic material to provideanti-CD20 antibodies, or antigen-binding fragments, variants, orderivatives thereof, of the invention, the polynucleotides encoding theanti-CD20 antibodies are typically inserted in an expression vector forintroduction into host cells that may be used to produce the desiredquantity of anti-CD20 antibody.

Recombinant expression of an antibody, or fragment, derivative or analogthereof, e.g., a heavy or light chain of an antibody that binds to atarget molecule described herein, e.g., CD20, requires construction ofan expression vector containing a polynucleotide that encodes theantibody. Once a polynucleotide encoding an antibody molecule or a heavyor light chain of an antibody, or portion thereof (preferably containingthe heavy or light chain variable domain), of the invention has beenobtained, the vector for the production of the antibody molecule may beproduced by recombinant DNA technology using techniques well known inthe art. Thus, methods for preparing a protein by expressing apolynucleotide containing an antibody encoding nucleotide sequence aredescribed herein. Methods that are well known to those skilled in theart can be used to construct expression vectors containing antibodycoding sequences and appropriate transcriptional and translationalcontrol signals. These methods include, for example, in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding an antibody molecule of theinvention, or a heavy or light chain thereof, or a heavy or light chainvariable domain, operably linked to a promoter. Such vectors may includethe nucleotide sequence encoding the constant region of the antibodymolecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of theantibody may be cloned into such a vector for expression of the entireheavy or light chain.

The term “vector” or “expression vector” is used herein to mean vectorsused in accordance with the present invention as a vehicle forintroducing into and expressing a desired gene in a host cell. As knownto those skilled in the art, such vectors may easily be selected fromthe group consisting of plasmids, phages, viruses and retroviruses. Ingeneral, vectors compatible with the instant invention will comprise aselection marker, appropriate restriction sites to facilitate cloning ofthe desired gene and the ability to enter and/or replicate in eukaryoticor prokaryotic cells.

For the purposes of this invention, numerous expression vector systemsmay be employed. For example, one class of vector utilizes DNA elementsthat are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses(RSV, MMTV or MOMLV) or SV40 virus. Others involve the use ofpolycistronic systems with internal ribosome binding sites.Additionally, cells that have integrated the DNA into their chromosomesmay be selected by introducing one or more markers which allow selectionof transfected host cells. The marker may provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation. Additional elements may also beneeded for optimal synthesis of mRNA. These elements may include signalsequences, splice signals, as well as transcriptional promoters,enhancers, and termination signals.

In particularly preferred embodiments, the cloned variable region genesare inserted into an expression vector along with the heavy and lightchain constant region genes (preferably human) synthesized as discussedabove. Of course, any expression vector that is capable of elicitingexpression in eukaryotic cells may be used in the present invention.Examples of suitable vectors include, but are not limited to plasmidspcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2,pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2 (available fromInvitrogen, San Diego, Calif.), and plasmid pCI (available from Promega,Madison, Wis.). In general, screening large numbers of transformed cellsfor those that express suitably high levels of immunoglobulin heavy andlight chains is routine experimentation that can be carried out, forexample, by robotic systems.

More generally, once the vector or DNA sequence encoding a monomericsubunit of the anti-CD20 antibody has been prepared, the expressionvector may be introduced into an appropriate host cell. Introduction ofthe plasmid into the host cell can be accomplished by various techniqueswell known to those of skill in the art. These include, but are notlimited to, transfection (including electroporation), protoplast fusion,calcium phosphate precipitation, cell fusion with enveloped DNA,microinjection, and infection with intact virus. See, Ridgway (1988)“Mammalian Expression Vectors” in Vectors, ed. Rodriguez and Denhardt(Butterworths, Boston, Mass.), Chapter 24.2, pp. 470-472. Typically,plasmid introduction into the host is via electroporation. The hostcells harboring the expression construct are grown under conditionsappropriate to the production of the light chains and heavy chains, andassayed for heavy and/or light chain protein synthesis. Exemplary assaytechniques include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), or fluorescence-activated cell sorter analysis(FACS), immunohistochemistry and the like.

The expression vector is transferred to a host cell by conventionaltechniques, and the transfected cells are then cultured by conventionaltechniques to produce an antibody for use in the methods describedherein. Thus, the invention includes host cells containing apolynucleotide encoding an antibody of the invention, or a heavy orlight chain thereof, operably linked to a heterologous promoter. Inpreferred embodiments for the expression of double-chained antibodies,vectors encoding both the heavy and light chains may be co-expressed inthe host cell for expression of the entire immunoglobulin molecule, asdetailed below.

As used herein, “host cells” refers to cells that harbor vectorsconstructed using recombinant DNA techniques and encoding at least oneheterologous gene. In descriptions of processes for isolation ofantibodies from recombinant hosts, the terms “cell” and “cell culture”are used interchangeably to denote the source of antibody unless it isclearly specified otherwise. In other words, recovery of polypeptidefrom the “cells” may mean either from spun down whole cells, or from thecell culture containing both the medium and the suspended cells.

A variety of host-expression vector systems may be utilized to expressantibody molecules for use in the methods described herein. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells that may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. These include, but are not limited to,microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing antibody coding sequences; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing antibody coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing antibody coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter). Preferably, bacterial cells such asEscherichia coli, and more preferably, eukaryotic cells, especially forthe expression of whole recombinant antibody molecule, are used for theexpression of a recombinant antibody molecule. For example, mammaliancells such as Chinese hamster ovary cells (CHO), in conjunction with avector such as the major intermediate early gene promoter element fromhuman cytomegalovirus is an effective expression system for antibodies(Foecking et al. (1986) Gene 45: 101; Cockett et al. (1990)Bio/Technology 8:2).

The host cell line used for protein expression is often of mammalianorigin; those skilled in the art are credited with ability topreferentially determine particular host cell lines that are best suitedfor the desired gene product to be expressed therein. Exemplary hostcell lines include, but are not limited to, CHO (Chinese Hamster Ovary),DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (humancervical carcinoma), CV1 (monkey kidney line), COS (a derivative of CV1with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, 293, WI38,R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK(hamster kidney line), SP2/O (mouse myeloma), P3x63-Ag3.653 (mousemyeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte)and 293 (human kidney). Host cell lines are typically available fromcommercial services, the American Tissue Culture Collection or frompublished literature.

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theantibody molecule may be engineered. Rather than using expressionvectors that contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which stably express theantibody molecule.

A number of selection systems may be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler et al. (1977) Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska andSzybalski (1992) Proc. Natl. Acad. Sci. USA 48:202), and adeninephosphoribosyltransferase (Lowy et al. (1980) Cell 22:817) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al. (1980) Natl. Acad. Sci. USA 77:357; O'Hare et al. (1981) Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg (1981) Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu (1991) Biotherapy 3:87-95;Tolstoshev (1993) Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan(1993) Science 260:926-932; and Morgan and Anderson (1993) Ann. Rev.Biochem. 62:191-217 (1993); TIB TECH 11(5):155-215 (May, 1993); andhygro, which confers resistance to hygromycin (Santerre et al. (1984)Gene 30:147. Methods commonly known in the art of recombinant DNAtechnology which can be used are described in Ausubel et al. (1993)Current Protocols in Molecular Biology (John Wiley & Sons, NY); Kriegler(1990) “Gene Transfer and Expression” in A Laboratory Manual (StocktonPress, NY); Dracopoli et al. (eds) (1994) Current Prolocols in HumanGenetics (John Wiley & Sons, N.Y.) Chapters 12 and 13; Colberre-Garapinet al. (1981) J. Mol. Biol. 150:1, which are incorporated by referenceherein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel (1987) “TheUse of Vectors Based on Gene Amplification for the Expression of ClonedGenes in Mammalian Cells in DNA Cloning” (Academic Press, NY) Vol. 3.When a marker in the vector system expressing antibody is amplifiable,an increase in the level of inhibitor present in culture of host cellwill increase the number of copies of the marker gene. Since theamplified region is associated with the antibody gene, production of theantibody will also increase (Crouse et al. (1983) Mol. Cell. Biol.3:257).

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-)affinity chromatography, e.g., afterpreferential biosynthesis of a synthetic hinge region polypeptide orprior to or subsequent to HIC chromatography.

Genes encoding anti-CD20 antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention can also be expressedby non-mammalian cells such as bacteria or yeast or plant cells.Bacteria that readily take up nucleic acids include members of theenterobacteriaceae, such as strains of Escherichia coli or Salmonella;Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the heterologous polypeptides typically becomepart of inclusion bodies. The heterologous polypeptides must beisolated, purified and then assembled into functional molecules. Wheretetravalent forms of antibodies are desired, the subunits will thenself-assemble into tetravalent antibodies (WO 02/096948A2).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al. (1983) EMBO J.2:1791), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye and Inouye (1985)Nucleic Acids Res. 13:3101-3109; Van Heeke and Schuster (1989) J. Biol.Chem. 24:5503-5509); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In addition to prokaryotes, eukaryotic microbes may also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available, e.g., Pichia pastoris.

For expression in Saccharomyces, the plasmid YRp7, for example,(Stinchcomb et al. (1979) Nature 282:39; Kingsman et al. (1979) Gene7:141; Tschemper et al. (1980) Gene 10:157) is commonly used. Thisplasmid already contains the TRP1 gene, which provides a selectionmarker for a mutant strain of yeast lacking the ability to grow intryptophan, for example ATCC No. 44076 or PEP4-1 (Jones (1977) Genetics85:12). The presence of the trp1 lesion as a characteristic of the yeasthost cell genome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is typically used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The antibody coding sequencemay be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.Alternatively, a preferred method for increasing the affinity ofantibodies of the invention is disclosed in U.S. Patent ApplicationPublication No. 2002 0123057 A1.

VII. Treatment Methods Using Therapeutic Anti-CD20 Antibodies

Methods of the invention are directed to the use of anti-CD20antibodies, including antigen-binding fragments, variants, andderivatives thereof, to treat patients having a disease associated withCD20-expressing cells. By “CD20-expressing cell” is intended normal andmalignant B cells expressing CD20 antigen. Methods for detecting CD20expression in cells are well known in the art and include, but are notlimited to, PCR techniques, immunohistochemistry, flow cytometry,Western blot, ELISA, and the like. By “malignant” B cell is intended anyneoplastic B cell, including but not limited to B cells derived fromlymphomas including low-, intermediate-, and high-grade B celllymphomas, immunoblastic lymphomas, non-Hodgkin's lymphomas, Hodgkin'sdisease, Epstein-Barr Virus (EBV) induced lymphomas, and AIDS-relatedlymphomas, as well as B cell acute lymphoblastic leukemias, myelomas,chronic lymphocytic leukemias, acute myeloblastic leukemias, and thelike.

Though the following discussion refers to diagnostic methods andtreatment of various diseases and disorders with an anti-CD20 antibodyof the invention, the methods described herein are also applicable tothe antigen-binding fragments, variants, and derivatives of theseanti-CD20 antibodies that retain the desired properties of the anti-CD20antibodies of the invention, i.e., capable of specifically binding CD20and having increased CDC activity and/or increased binding affinity forthe CD20 antigen.

“Treatment” is herein defined as the application or administration of ananti-CD20 antibody to a patient, or application or administration of ananti-CD20 antibody to an isolated tissue or cell line from a patient,where the patient has a disease, a symptom of a disease, or apredisposition toward a disease, where the purpose is to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve, or affect thedisease, the symptoms of the disease, or the predisposition toward thedisease. By “treatment” is also intended the application oradministration of a pharmaceutical composition comprising the anti-CD20antibody to a patient, or application or administration of apharmaceutical composition comprising the anti-CD20 antibody to anisolated tissue or cell line from a patient, who has a disease, asymptom of a disease, or a predisposition toward a disease, where thepurpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve, or affect the disease, the symptoms of the disease, or thepredisposition toward the disease.

By “anti-tumor activity” is intended a reduction in the rate ofmalignant CD20-expressing cell proliferation or accumulation, and hencea decline in growth rate of an existing tumor or in a tumor that arisesduring therapy, and/or destruction of existing neoplastic (tumor) cellsor newly formed neoplastic cells, and hence a decrease in the overallsize of a tumor during therapy. By “anti-inflammatory activity” isintended a reduction or prevention of inflammation. Therapy with atleast one anti-CD20 antibody causes a physiological response that isbeneficial with respect to treatment of disease states associated withCD20-expressing cells in a human.

In this manner, the methods of the invention find use in the treatmentof non-Hodgkin's lymphomas related to abnormal, uncontrollable B cellproliferation or accumulation. For purposes of the present invention,such lymphomas will be referred to according to the Working Formulationclassification scheme, that is those B cell lymphomas categorized as lowgrade, intermediate grade, and high grade (see “The Non-Hodgkin'sLymphoma Pathologic Classification Project” in Cancer 49:2112-2135(1982)). Thus, low-grade B cell lymphomas include small lymphocytic,follicular small-cleaved cell, and follicular mixed small-cleaved andlarge cell lymphomas; intermediate-grade lymphomas include follicularlarge cell, diffuse small cleaved cell, diffuse mixed small and largecell, and diffuse large cell lymphomas; and high-grade lymphomas includelarge cell immunoblastic, lymphoblastic, and small non-cleaved celllymphomas of the Burkitt's and non-Burkitt's type.

It is recognized that the methods of the invention are useful in thetherapeutic treatment of B cell lymphomas that are classified accordingto the Revised European and American Lymphoma Classification (REAL)system. Such B cell lymphomas include, but are not limited to, lymphomasclassified as precursor B cell neoplasms, such as B lymphoblasticleukemia/lymphoma; peripheral B cell neoplasms, including B cell chroniclymphocytic leukemia/small lymphocytic lymphoma, lymphoplasmacytoidlymphoma/immunocytoma, mantle cell lymphoma (MCL), follicle centerlymphoma (follicular) (including diffuse small cell, diffuse mixed smalland large cell, and diffuse large cell lymphomas), marginal zone B celllymphoma (including extranodal, nodal, and splenic types), hairy cellleukemia, plasmacytoma/myeloma, diffuse large cell B cell lymphoma ofthe subtype primary mediastinal (thymic), Burkitt's lymphoma, andBurkitt's like high grade B cell lymphoma; acute leukemias; acutelymphocytic leukemias; myeloblastic leukemias; acute myelocyticleukemias; promyelocytic leukemia; myelomonocytic leukemia; monocyticleukemia; erythroleukemia; granulocytic leukemia (chronic myelocyticleukemia); chronic lymphocytic leukemia; polycythemia vera; multiplemyeloma; Waldenstrom's macroglobulinemia; heavy chain disease; andunclassifiable low-grade or high-grade B cell lymphomas.

It is recognized that the methods of the invention may be useful inpreventing further tumor outgrowths arising during therapy. The methodsof the invention are particularly useful in the treatment of subjectshaving low-grade B cell lymphomas, particularly those subjects havingrelapses following standard chemotherapy. Low-grade B cell lymphomas aremore indolent than the intermediate- and high-grade B cell lymphomas andare characterized by a relapsing/remitting course. Thus, treatment ofthese lymphomas is improved using the methods of the invention, asrelapse episodes are reduced in number and severity.

In accordance with the methods of the present invention, at least oneanti-CD20 antibody as defined elsewhere herein is used to promote apositive therapeutic response with respect to a malignant human B cell.By “positive therapeutic response” with respect to cancer treatment isintended an improvement in the disease in association with theanti-tumor activity of these antibodies or fragments thereof, and/or animprovement in the symptoms associated with the disease. That is, ananti-proliferative effect, the prevention of further tumor outgrowths, areduction in tumor size, a reduction in the number of cancer cells,and/or a decrease in one or more symptoms associated with the diseasecan be observed. Thus, for example, an improvement in the disease may becharacterized as a complete response. By “complete response” is intendedan absence of clinically detectable disease with normalization of anypreviously abnormal radiographic studies, bone marrow, and cerebrospinalfluid (CSF). Such a response must persist for at least one monthfollowing treatment according to the methods of the invention.Alternatively, an improvement in the disease may be categorized as beinga partial response. By “partial response” is intended at least about a50% decrease in all measurable tumor burden (i.e., the number of tumorcells present in the subject) in the absence of new lesions andpersisting for at least one month. Such a response is applicable tomeasurable tumors only.

Tumor response can be assessed for changes in tumor morphology (i.e.,overall tumor burden, tumor size, and the like) using screeningtechniques such as magnetic resonance imaging (MRI) scan, x-radiographicimaging, computed tomographic (CT) scan, bioluminescent imaging, forexample, luciferase imaging, bone scan imaging, and tumor biopsysampling including bone marrow aspiration (BMA). In addition to thesepositive therapeutic responses, the subject undergoing therapy with theanti-CD20 antibody may experience the beneficial effect of animprovement in the symptoms associated with the disease. Thus for B celltumors, the subject may experience a decrease in the so-called Bsymptoms, i.e., night sweats, fever, weight loss, and/or urticaria.

The anti-CD20 antibodies described herein may also find use in thetreatment of inflammatory diseases and deficiencies or disorders of theimmune system that are associated with CD-20 expressing cells,including, but not limited to, systemic lupus erythematosus, psoriasis,scleroderma, CREST syndrome, inflammatory myositis, Sjogren's syndrome,mixed connective tissue disease, rheumatoid arthritis, multiplesclerosis, inflammatory bowel disease, acute respiratory distresssyndrome, pulmonary inflammation, idiopathic pulmonary fibrosis,osteoporosis, delayed type hypersensitivity, asthma, primary biliarycirrhosis, and idiopathic thrombocytopenic purpura.

Inflammatory diseases are characterized by inflammation and tissuedestruction, or a combination thereof. “Inflammatory disease” includesany inflammatory immune-mediated process where the initiating event ortarget of the immune response involves non-self antigen(s), including,for example, alloantigens, xenoantigens, viral antigens, bacterialantigens, unknown antigens, or allergens.

Further, for purposes of the present invention, the term “inflammatorydisease(s)” includes “autoimmune disease(s).” As used herein, the term“autoimmunity” is generally understood to encompass inflammatoryimmune-mediated processes involving “self” antigens. In autoimmunediseases, self antigen(s) trigger host immune responses.

Also, the present invention includes treatment of inflammationassociated with tissue transplant rejection. “Transplant rejection” or“graft rejection” refers to any host-mounted immune response against agraft including but not limited to HLA antigens, blood group antigens,and the like.

The invention can also be used to treat graft versus host disease, suchas that associated with bone marrow transplantation, for example. Insuch graft versus host disease, the donor bone marrow includeslymphocytes and cells that mature into lymphocytes. The donor'slymphocytes recognize the recipient's antigens as non-self and mount aninflammatory immune response. Hence, as used herein, “graft versus hostdisease” or “graft versus host reaction” refers to any T cell mediatedimmune response in which donor lymphocytes react to the host's antigens.

The anti-CD20 described herein can be used in accordance with themethods of the invention to treat autoimmune and/or inflammatorydisorders including, but not limited to, systemic lupus erythematosus(SLE), discoid lupus, lupus nephritis, sarcoidosis, inflammatoryarthritis, including juvenile arthritis, rheumatoid arthritis, psoriaticarthritis, Reiter's syndrome, ankylosing spondylitis, and goutyarthritis, rejection of an organ or tissue transplant, hyperacute,acute, or chronic rejection and/or graft versus host disease, multiplesclerosis, hyper IgE syndrome, polyarteritis nodosa, primary biliarycirrhosis, inflammatory bowel disease, Crohn's disease, celiac's disease(gluten-sensitive enteropathy), autoimmune hepatitis, pernicious anemia,autoimmune hemolytic anemia, psoriasis, scleroderma, myasthenia gravis,autoimmune thrombocytopenic purpura, autoimmune thyroiditis, Grave'sdisease, Hashimoto's thyroiditis, immune complex disease, chronicfatigue immune dysfunction syndrome (CFIDS), polymyositis anddermatomyositis, cryoglobulinemia, thrombolysis, cardiomyopathy,pemphigus vulgaris, pulmonary interstitial fibrosis, Type I and Type IIdiabetes mellitus, type 1, 2, 3, and 4 delayed-type hypersensitivity,allergy or allergic disorders, unwanted/unintended immune responses totherapeutic proteins (see for example, U.S. Patent Application No. US2002/0119151 and Koren, et al. (2002) Curr. Pharm. Biotechnol.3:349-60), asthma, Churg-Strauss syndrome (allergic granulomatosis),atopic dermatitis, allergic and irritant contact dermatitis, urtecaria,IgE-mediated allergy, atherosclerosis, vasculitis, idiopathicinflammatory myopathies, hemolytic disease, Alzheimer's disease, chronicinflammatory demyelinating polyneuropathy, and the like. In some otherembodiments, the anti-CD20 antibodies of the invention are useful intreating pulmonary inflammation including but not limited to lung graftrejection, asthma, sarcoidosis, emphysema, cystic fibrosis, idiopathicpulmonary fibrosis, chronic bronchitis, allergic rhinitis and allergicdiseases of the lung such as hypersensitivity pneumonitis, eosinophilicpneumonia, bronchiolitis obliterans due to bone marrow and/or lungtransplantation or other causes, graft atherosclerosis/graftphlebosclerosis, as well as pulmonary fibrosis resulting from collagen,vascular, and autoimmune diseases such as rheumatoid arthritis and lupuserythematosus.

In accordance with the methods of the present invention, at least oneanti-CD20 antibody as defined elsewhere herein is used to promote apositive therapeutic response with respect to treatment or prevention ofan autoimmune disease and/or inflammatory disease. By “positivetherapeutic response” with respect to an autoimmune disease and/orinflammatory disease is intended an improvement in the disease inassociation with the anti-inflammatory activity of these antibodies,and/or an improvement in the symptoms associated with the disease. Thatis, an anti-proliferative effect, the prevention of furtherproliferation of the CD20-expressing cell, a reduction in theinflammatory response including but not limited to reduced secretion ofinflammatory cytokines, adhesion molecules, proteases, immunoglobulins(in instances where the CD20 bearing cell is a B cell), combinationsthereof, and the like, increased production of anti-inflammatoryproteins, a reduction in the number of autoreactive cells, an increasein immune tolerance, inhibition of autoreactive cell survival, and/or adecrease in one or more symptoms mediated by stimulation ofCD20-expressing cells can be observed. Such positive therapeuticresponses are not limited to the route of administration and maycomprise administration to the donor, the donor tissue (such as forexample organ perfusion), the host, any combination thereof, and thelike.

Clinical response can be assessed using screening techniques such asmagnetic resonance imaging (MRI) scan, x-radiographic imaging, computedtomographic (CT) scan, flow cytometry or fluorescence-activated cellsorter (FACS) analysis, histology, gross pathology, and blood chemistry,including but not limited to changes detectable by ELISA, RIA,chromatography, and the like. In addition to these positive therapeuticresponses, the subject undergoing therapy with the anti-CD20 antibody orantigen-binding fragment thereof may experience the beneficial effect ofan improvement in the symptoms associated with the disease.

By “therapeutically effective dose or amount” or “effective amount” isintended an amount of anti-CD20 that when administered brings about apositive therapeutic response with respect to treatment of a patientwith a disease associated with CD20-expressing cells. In someembodiments of the invention, a therapeutically effective dose of theanti-CD20 antibody is in the range from about 0.01 mg/kg to about 40mg/kg, from about 0.01 mg/kg to about 30 mg/kg, from about 0.1 mg/kg toabout 30 mg/kg, from about 1 mg/kg to about 30 mg/kg, from about 3 mg/kgto about 30 mg/kg, from about 3 mg/kg to about 25 mg/kg, from about 3mg/kg to about 20 mg/kg, from about 5 mg/kg to about 15 mg/kg, or fromabout 7 mg/kg to about 12 mg/kg. It is recognized that the method oftreatment may comprise a single administration of a therapeuticallyeffective dose or multiple administrations of a therapeuticallyeffective dose of the anti-CD20 antibody.

The anti-CD20 antibodies can be used in combination with knownchemotherapeutics and cytokines for the treatment of disease statescomprising CD20-expressing cells. For example, the anti-CD20 antibodiesof the invention can be used in combination with cytokines such asinterleukin-2. In another embodiment, the anti-CD20 antibodies of theinvention can be used in combination with rituximab (IDEC-C2B8;Rituxan®; IDEC Pharmaceuticals Corp., San Diego, Calif.).

In this manner, the anti-CD20 antibodies described herein areadministered in combination with at least one other cancer therapy,including, but not limited to, surgery or surgical procedures (e.g.splenectomy, hepatectomy, lymphadenectomy, leukophoresis, bone marrowtransplantation, and the like); radiation therapy; chemotherapy,optionally in combination with autologous bone marrow transplant, wheresuitable chemotherapeutic agents include, but are not limited to,fludarabine or fludarabine phosphate, chlorambucil, vincristine,pentostatin, 2-chlorodeoxyadenosine (cladribine), cyclophosphamide,doxorubicin, prednisone, and combinations thereof, for example,anthracycline-containing regimens such as CAP (cyclophosphamide,doxorubicin plus prednisone), CHOP (cyclophosphamide, vincristine,prednisone plus doxorubicin), VAD (vincritsine, doxorubicin, plusdexamethasone), MP (melphalan plus prednisone), and other cytotoxicand/or therapeutic agents used in chemotherapy such as mitoxantrone,daunorubicin, idarubicin, asparaginase, and antimetabolites, including,but not limited to, cytarabine, methotrexate, 5-fluorouracildecarbazine, 6-thioguanine, 6-mercaptopurine, and nelarabine; otheranti-cancer monoclonal antibody therapy (for example, alemtuzumab(Campath®) or other anti-CD52 antibody targeting the CD52 cell-surfaceglycoprotein on malignant B cells; rituximab (Rituxan®), the fully humanantibody HuMax-CD20, R-1594, IMMU-106, TRU-015, AME-133,tositumomab/I-131 tositumomab (Bexxar®), ibritumomab tiuxetan(Zevalin®), or any other therapeutic anti-CD20 antibody targeting theCD20 antigen on malignant B cells; anti-CD19 antibody (for example,MT103, a bispecific antibody); anti-CD22 antibody (for example, thehumanized monoclonal antibody epratuzumab); bevacizumab (Avastin®) orother anti-cancer antibody targeting human vascular endothelial growthfactor; anti-CD22 antibody targeting the CD22 antigen on malignant Bcells (for example, the monoclonal antibody BL-22, an alphaCD22 toxin);α-M-CSF antibody targeting macrophage colony stimulating factor;antibodies targeting the receptor activator of nuclear factor-kappaB(RANK) and its ligand (RANKL), which are overexpressed in multiplemyeloma; anti-CD23 antibody targeting the CD23 antigen on malignant Bcells (for example, IDEC-152); anti-CD80 antibody targeting the CD80antigen (for example, IDEC-114); anti-CD38 antibody targeting the CD38antigen on malignant B cells; antibodies targeting majorhistocompatibility complex class II receptors (anti-MHC antibodies)expressed on malignant B cells; anti-CD40 antibodies (for example,SGN-40) targeting the CD40 antigen on malignant B cells; and antibodiestargeting tumor necrosis factor-related apoptosis-inducing ligandreceptor 1 (TRAIL-R1) (for example, the agonistic human monoclonalantibody HGS-ETR1) and TRAIL-R2 expressed on a number of solid tumorsand tumors of hematopoietic origin); small molecule-based cancertherapy, including, but not limited to, microtubule and/or topoisomeraseinhibitors (for example, the mitotic inhibitor dolastatin and dolastatinanalogues; the tubulin-binding agent T900607; XL119; and thetopoisomerase inhibitor aminocamptothecin), SDX-105 (bendamustinehydrochloride), ixabepilone (an epothilone analog, also referred to asBMS-247550), protein kinase C inhibitors, for example, midostaurin((PKC-412, CGP 41251, N-benzoylstaurosporine), pixantrone, eloxatin (anantineoplastic agent), ganite (gallium nitrate), Thalomid®(thalidomide), immunomodulatory derivatives of thalidomide (for example,revlimid (formerly revimid)), Affinitak™ (antisense inhibitor of proteinkinase C-alpha), SDX-101 (R-etodolac, inducing apoptosis of malignantlymphocytes), second-generation purine nucleoside analogs such asclofarabine, inhibitors of production of the protein Bcl-2 by cancercells (for example, the antisense agents oblimersen and Genasense®),proteasome inhibitors (for example, Velcade™ (bortezomib)), smallmolecule kinase inhibitors (for example, CHIR-258), small molecule VEGFinhibitors (for example, ZD-6474), small molecule inhibitors of heatshock protein (HSP) 90 (for example, 17-AAG), small molecule inhibitorsof histone deacetylases (for example, hybrid/polar cytodifferentiationHPC) agents such as suberanilohydroxamic acid (SAHA), and FR-901228) andapoptotic agents such as Trisenox® (arsenic trioxide) and Xcytrin®(motexafin gadolinium); vaccine/immunotherapy-based cancer therapies,including, but not limited to, vaccine approaches (for example, Id-KLH,oncophage, vitalethine), personalized immunotherapy or active idiotypeimmunotherapy (for example, MyVax® Personalized Immunotherapy, formallydesignated GTOP-99), Promune® (CpG 7909, a synthetic agonist fortoll-like receptor 9 (TLR9)), interferon-alpha therapy, interleukin-2(IL-2) therapy, IL-12 therapy, IL-15 therapy, and IL-21 therapy; steroidtherapy; or other cancer therapy; where the additional cancer therapy isadministered prior to, during, or subsequent to the anti-CD20 antibodytherapy.

Thus, where the combined therapies comprise administration of ananti-CD20 antibody in combination with administration of anothertherapeutic agent, as with chemotherapy, radiation therapy, otheranti-cancer antibody therapy, small molecule-based cancer therapy, orvaccine/immunotherapy-based cancer therapy, the methods of the inventionencompass coadministration, using separate formulations or a singlepharmaceutical formulation, and consecutive administration in eitherorder. Where the methods of the present invention comprise combinedtherapeutic regimens, these therapies can be given simultaneously, i.e.,the anti-CD20 antibody is administered concurrently or within the sametime frame as the other cancer therapy (i.e., the therapies are going onconcurrently, but the anti-CD20 antibody is not administered preciselyat the same time as the other cancer therapy). Alternatively, theanti-CD20 antibody of the present invention may also be administeredprior to or subsequent to the other cancer therapy. Sequentialadministration of the different cancer therapies may be performedregardless of whether the treated subject responds to the first courseof therapy to decrease the possibility of remission or relapse. Wherethe combined therapies comprise administration of the anti-CD20 antibodyin combination with administration of a cytotoxic agent, preferably theanti-CD20 antibody is administered prior to administering the cytotoxicagent.

In some embodiments of the invention, the anti-CD20 antibodies describedherein are administered in combination with chemotherapy, and optionallyin combination with autologous bone marrow transplantation, wherein theantibody and the chemotherapeutic agent(s) may be administeredsequentially, in either order, or simultaneously (i.e., concurrently orwithin the same time frame). Examples of suitable chemotherapeuticagents include, but are not limited to, fludarabine or fludarabinephosphate, chlorambucil, vincristine, pentostatin,2-chlorodeoxyadenosine (cladribine), cyclophosphamide, doxorubicin,prednisone, and combinations thereof, for example,anthracycline-containing regimens such as CAP (cyclophosphamide,doxorubicin plus prednisone), CHOP (cyclophosphamide, vincristine,prednisone plus doxorubicin), VAD (vincritsine, doxorubicin, plusdexamethasone), MP (melphalan plus prednisone), and other cytotoxicand/or therapeutic agents used in chemotherapy such as mitoxantrone,daunorubicin, idarubicin, asparaginase, and antimetabolites, including,but not limited to, cytarabine, methotrexate, 5-fluorouracildecarbazine, 6-thioguanine, 6-mercaptopurine, and nelarabine. In someembodiments, the anti-CD20 antibody is administered prior to treatmentwith the chemotherapeutic agent. In alternative embodiments, theanti-CD20 antibody is administered after treatment with thechemotherapeutic agent. In yet other embodiments, the chemotherapeuticagent is administered simultaneously with the anti-CD20 antibody.

Thus, for example, in some embodiments, the anti-CD20 antibody isadministered in combination with fludarabine or fludarabine phosphate.In one such embodiment, the anti-CD20 antibody is administered prior toadministration of fludarabine or fludarabine phosphate. In alternativeembodiments, the anti-CD20 antibody is administered after treatment withfludarabine or fludarabine phosphate. In yet other embodiments, thefludarabine or fludarabine phosphate is administered simultaneously withthe anti-CD20 antibody.

In other embodiments of the invention, chlorambucil, an alkylating drug,is administered in combination with an anti-CD20 antibody describedherein. In one such embodiment, the anti-CD20 antibody is administeredprior to administration of chlorambucil. In alternative embodiments, theanti-CD20 antibody is administered after treatment with chlorambucil. Inyet other embodiments, the chlorambucil is administered simultaneouslywith the anti-CD20 antibody.

In yet other embodiments, anthracycline-containing regimens such as CAP(cyclophosphamide, doxorubicin plus prednisone) and CHOP(cyclophosphamide, vincristine, prednisone plus doxorubicin) may becombined with administration of an anti-CD20 antibody described herein.In one such embodiment, the anti-CD20 antibody is administered prior toadministration of anthracycline-containing regimens. In otherembodiments, the anti-CD20 antibody is administered after treatment withanthracycline-containing regimens. In yet other embodiments, theanthracycline-containing regimen is administered simultaneously with theanti-CD20 antibody.

In alternative embodiments, an anti-CD20 antibody described herein isadministered in combination with alemtuzumab (Campath®; distributed byBerlex Laboratories, Richmond, Calif.). Alemtuzumab is a recombinanthumanized monoclonal antibody (Campath-1H) that targets the CD52 antigenexpressed on malignant B cells. In one such embodiment, the anti-CD20antibody is administered prior to administration of alemtuzumab. Inother embodiments, the anti-CD20 antibody is administered aftertreatment with alemtuzumab. In yet other embodiments, the alemtuzumab isadministered simultaneously with the anti-CD20 antibody.

In alternative embodiments, an anti-CD20 antibody described herein isadministered in combination with another therapeutic anti-CD20 antibodytargeting the CD20 antigen on malignant B cells, for example, rituximab(Rituxan®), the fully human antibody HuMax-CD20, R-1594, IMMU-106,TRU-015, AME-133, tositumomab/1-131 tositumomab (Bexxar®), oribritumomab tiuxetan (Zevalin®). In one such embodiment, the anti-CD20antibody of the invention is administered prior to administration of theother anti-CD20 antibody. In other embodiments, the anti-CD20 antibodyof the invention is administered after treatment with the otheranti-CD20 antibody. In yet other embodiments, the anti-CD20 antibody ofthe invention is administered simultaneously with the other anti-CD20antibody.

In alternative embodiments, an anti-CD20 antibody described herein isadministered in combination with a small molecule-based cancer therapy,including, but not limited to, microtubule and/or topoisomeraseinhibitors (for example, the mitotic inhibitor dolastatin and dolastatinanalogues; the tubulin-binding agent T900607; XL119; and thetopoisomerase inhibitor aminocamptothecin), SDX-105 (bendamustinehydrochloride), ixabepilone (an epothilone analog, also referred to asBMS-247550), protein kinase C inhibitors, for example, midostaurin((PKC-412, CGP 41251, N-benzoylstaurosporine), pixantrone, eloxatin (anantineoplastic agent), ganite (gallium nitrate), Thalomid®(thalidomide), immunomodulatory derivatives of thalidomide (for example,revlimid (formerly revimid)), Affinitak™ (antisense inhibitor of proteinkinase C-alpha), SDX-101 (R-etodolac, inducing apoptosis of malignantlymphocytes), second-generation purine nucleoside analogs such asclofarabine, inhibitors of production of the protein Bcl-2 by cancercells (for example, the antisense agents oblimersen and Genasense®),proteasome inhibitors (for example, Velcade™ (bortezomib)), smallmolecule kinase inhibitors (for example, CHIR-258), small molecule VEGFinhibitors (for example, ZD-6474), small molecule inhibitors of heatshock protein (HSP) 90 (for example, 17-AAG), small molecule inhibitorsof histone deacetylases (for example, hybrid/polar cytodifferentiationHPC) agents such as suberanilohydroxamic acid (SAHA), and FR-901228) andapoptotic agents such as Trisenox® (arsenic trioxide) and Xcytrin®(motexafin gadolinium). In one such embodiment, the anti-CD20 antibodyis administered prior to administration of the small molecule-basedcancer therapy. In other embodiments, the anti-CD20 antibody isadministered after treatment with the small molecule-based cancertherapy. In yet other embodiments, the small molecule-based cancertherapy is administered simultaneously with the anti-CD20 antibody.

In yet other embodiments, an anti-CD20 antibody described herein can beused in combination with vaccine/immunotherapy-based cancer therapy,including, but not limited to, vaccine approaches (for example, Id-KLH,oncophage, vitalethine), personalized immunotherapy or active idiotypeimmunotherapy (for example, MyVax® Personalized Immunotherapy, formallydesignated GTOP-99), Promune® (CpG 7909, a synthetic agonist fortoll-like receptor 9 (TLR9)), interferon-alpha therapy, interleukin-2(IL-2) therapy, IL-12 therapy, IL-15 therapy, or IL-21 therapy; orsteroid therapy. In one such embodiment, the anti-CD20 antibody isadministered prior to administration of the vaccine/immunotherapy-basedcancer therapy. In other embodiments, the anti-CD20 antibody isadministered after treatment with the vaccine/immunotherapy-based cancertherapy. In yet other embodiments, the vaccine/immunotherapy-basedcancer therapy is administered simultaneously with the anti-CD20antibody.

In one such embodiment, an anti-CD20 antibody described herein can beused in combination with IL-2. IL-2, an agent known to expand the numberof natural killer (NK) effector cells in treated patients, can beadministered prior to, or concomitantly with, the anti-CD20 antibody ofthe invention. This expanded number of NK effector cells may lead toenhanced ADCC activity of the administered anti-CD20 antibody. In otherembodiments, IL-21 serves as the immunotherapeutic agent to stimulate NKcell activity when administered in combination with an anti-CD20antibody described herein.

The anti-CD20 antibodies of the invention can be used in combinationwith any known therapies for autoimmune and inflammatory diseases,including any agent or combination of agents that are known to beuseful, or which have been used or are currently in use, for treatmentof autoimmune and inflammatory diseases. Such therapies and therapeuticagents include, but are not limited to, surgery or surgical procedures(e.g., splenectomy, lymphadenectomy, thyroidectomy, plasmaphoresis,leukophoresis, cell, tissue, or organ transplantation, intestinalprocedures, organ perfusion, and the like), radiation therapy, therapysuch as steroid therapy and non-steroidal therapy, hormone therapy,cytokine therapy, therapy with dermatological agents (for example,topical agents used to treat skin conditions such as allergies, contactdermatitis, and psoriasis), immunosuppressive therapy, and otheranti-inflammatory monoclonal antibody therapy, and the like. In thismanner, the anti-CD20 antibodies described herein are administered incombination with at least one other therapy, including, but not limitedto, surgery, organ perfusion, radiation therapy, steroid therapy,non-steroidal therapy, antibiotic therapy, antifungal therapy, hormonetherapy, cytokine therapy, therapy with dermatological agents (forexample, topical agents used to treat skin conditions such as allergies,contact dermatitis, and psoriasis), immunosuppressive therapy, otheranti-inflammatory monoclonal antibody therapy, combinations thereof, andthe like. Thus, where the combined therapies comprise administration ofan anti-CD20 antibody in combination with administration of anothertherapeutic agent, as with steroids as one example, the methods of theinvention encompass coadministration, using separate formulations or asingle pharmaceutical formulation, and consecutive administration ineither order.

Where the methods of the present invention comprise combined therapeuticregimens, these therapies can be given simultaneously, i.e., theanti-CD20 antibody is administered concurrently or within the same timeframe as the other therapy (i.e., the therapies are going onconcurrently, but the anti-CD20 antibody is not administered preciselyat the same time as the other therapy). Alternatively, the anti-CD20antibody of the present invention may also be administered prior to orsubsequent to the other therapy. Sequential administration of thedifferent therapies may be performed regardless of whether the treatedsubject responds to the first course of therapy to decrease thepossibility of remission or relapse.

In some embodiments of the invention, the anti-CD20 antibodies describedherein are administered in combination with immunosuppressive drugs oranti-inflammatory drugs, wherein the antibody and the therapeuticagent(s) may be administered sequentially, in either order, orsimultaneously (i.e., concurrently or within the same time frame).Examples of suitable immunosuppressive drugs that can be administered incombination with the anti-CD20 antibodies of the invention include, butare not limited to, methotrexate, cyclophosphamide, mizoribine,chlorambucil, cyclosporine, such as, for example, aerosolizedcyclosporine (see, U.S. Patent Application Publication No.US20020006901, herein incorporated by reference in its entirety),tacrolimus (FK506; ProGraf™), mycophenolate mofetil, and azathioprine(6-mercaptopurine), sirolimus (rapamycin), deoxyspergualin, leflunomideand its malononitriloamide analogs; and immunosuppressive proteins,including, for example, anti-CTLA4 antibodies and Ig fusions, anti-Blymphocyte stimulator antibodies (e.g., LYMPHOSTAT-B™) and Ig fusions(BLyS-Ig), anti-CD80 antibodies and etanercept (Enbrel®), as well asanti-T cell antibodies such as anti-CD3 (OKT3), anti-CD4, and the like.Examples of suitable anti-inflammatory agents include, but are notlimited to, corticosteroids such as, for example, clobetasol,halobetasol, hydrocortisone, triamcinolone, betamethasone, fluocinole,fluocinonide, prednisone, prednisolone, methylprednisolone;non-steroidal anti-inflammatory drugs (NSAIDs) such as, for example,sulfasalazine, medications containing mesalamine (known as 5-ASAagents), celecoxib, diclofenac, etodolac, fenprofen, flurbiprofen,ibuprofen, ketoprofen, meclofamate, meloxicam, nabumetone, naproxen,oxaprozin, piroxicam, rofecoxib, salicylates, sulindac, and tolmetin;anti-inflammatory antibodies such as adalimumab (HUMIRA®, a TNF-αantagonist) and infliximab (Remicade®, a TNF-α antagonist), and thelike.

Transplant rejection and graft versus host disease can be hyperacute(humoral), acute (T cell mediated), or chronic (unknown etiology), or acombination thereof. Thus, the anti-CD20 antibodies of the invention areused in some embodiments to prevent and/or ameliorate rejection and/orsymptoms associated with hyperacute, acute, and/or chronic transplantrejection of any tissue, including, but not limited to, liver, kidney,pancreas, pancreatic islet cells, small intestine, lung, heart, corneas,skin, blood vessels, bone, heterologous or autologous bone marrow, andthe like. Graft tissues may be obtained from any donor and transplantedinto any recipient host, and thus the transplant procedure may comprisetransplanting animal tissue to humans (e.g., xenografts), transplantingtissue from one human to another human (e.g., allografts), and/ortransplanting tissue from one part of a human's body to another (e.g.,autografts). Treatment with the antibodies of the invention may alsoreduce transplantation sequelae such as fever, anorexia, hemodynamicabnormalities, leukopenia, white cell infiltration of the transplantedorgan/tissue, as well as opportunistic infections.

In some embodiments, the anti-CD20 antibodies of the invention may beused alone or in combination with immunosuppressive drugs to treatand/or prevent transplant rejection such as hyperacute, acute, and/orchronic rejection and/or graft versus host disease. Thus, in someembodiments where the anti-CD20 antibodies of the invention are used totreat graft rejection, the antibodies may used in combination withsuitable immunosuppressive drugs, including, but not limited, tomethotrexate; cyclophosphamide; mizoribine; chlorambucil; cyclosporine,such as, for example, aerosolized cyclosporine (see, U.S. PatentApplication Publication No. US20020006901, herein incorporated byreference in its entirety), tacrolimus (FK506; ProGraf™), mycophenolatemofetil, and azathioprine (6-mercaptopurine), sirolimus (rapamycin),deoxyspergualin, leflunomide and its malononitriloamide analogs; andimmunosuppressive proteins, including, for example, anti-CTLA antibodiesand Ig fusions, anti-B lymphocyte stimulator antibodies (e.g.,LYMPHOSTAT-B™) and Ig fusions (BLyS-Ig), anti-CD80 antibodies andetanercept (Enbrel®), as well as anti-T cell antibodies such as anti-CD3(OKT3), anti-CD4, and the like.

As such, it is specifically contemplated that the compositions andmethods of the invention are used in combination with other drugs tofurther improve symptoms and outcomes in transplant recipients, such asthose receiving lung grafts, for example. Thus, in some embodiments, theanti-CD20 antibodies of the invention are used to treat transplantrejection (such as, for example hyperacute, acute, and/or chronicrejection or graft versus host disease in lung transplant recipients)alone or in combination with parenterally and/or non-parenterallyadministered cyclosporine, including for example oral cyclosporine,injectable cyclosporine, aerosolized (e.g., inhaled) cyclosporine, andcombinations thereof. In some embodiments where at least a component ofthe therapy is aerosolized cyclosporine, the cyclosporine is deliveredto the lung of the recipient by inhalation of cyclosporine in aerosolspray form using, for example, a pressurized delivery device ornebulizer. The cyclosporine may be administered in either dry powder orwet form.

In some other embodiments, the anti-CD20 antibodies of the invention maybe used alone or in combination with immunosuppressive drugs to treatand/or prevent rheumatoid arthritis. Thus in some embodiments where theanti-CD20 antibodies of the invention are used to treat rheumatoidarthritis, the antibodies may used in combination with suitableimmunosuppressive drugs, including, but not limited to, methotrexate,cyclophosphamide, mizoribine, chlorambucil, cyclosporine, tacrolimus(FK506; PROGRAF™), mycophenolate mofetil, and azathioprine(6-mercaptopurine), sirolimus (rapamycin), deoxyspergualin, leflunomideand its malononitriloamide analogs; and immunosuppressive proteins,including, for example, anti-CTLA antibodies and Ig fusions, anti-Blymphocyte stimulator antibodies (e.g., LYMPHOSTAT-B™) and Ig fusions(BLyS-Ig), other anti-CD20 antibodies (e.g. RITUXAN®); the fully humanantibody HuMax-CD20, R-1594, IMMU-106, TRU-015, AME-133,tositumomab/I-131, tositumomab (Bexxar®), ibritumomab tituxetan(Zevalin®); anti-CD80 antibodies, and etanercept (ENBREL®), as well asanti-T cell antibodies such as anti-CD3 (OKT3), anti-CD4, and the like.As discussed above, treatment effectiveness may be assessed using anymeans and includes, but is not limited to, effectiveness as measured byclinical responses defined by the American College of Rheumatologycriteria, the European League of Rheumatism criteria, or any othercriteria. See for example, Felson et al. (1995) Arthritis. Rheum.38:727-35 and van Gestel et al. (1996) Arthritis Rheum. 39:34-40.

In yet other embodiments, the anti-CD20 antibodies of the invention maybe used alone or in combination with immunosuppressive drugs to treatand/or prevent multiple sclerosis. Thus in some embodiments where theanti-CD20 antibodies of the invention are used to treat multiplesclerosis, the antibodies may be used in combination with suitableimmunosuppressive drugs, including, but not limited to, methotrexate,cyclophosphamide, mizoribine, chlorambucil, cyclosporine, tacrolimus(FK506; PROGRAF™), mycophenolate mofetil, and azathioprine(6-mercaptopurine), sirolimus (rapamycin), deoxyspergualin, leflunomideand its malononitriloamide analogs; and immunosuppressive proteins,including, for example, anti-CTLA antibodies and Ig fusions, anti-Blymphocyte stimulator antibodies (e.g., LYMPHOSTAT-B™) and Ig fusions(BLyS-Ig), other anti-CD20 antibodies (e.g., RITUXAN®); the fully humanantibody HuMax-CD20, R-1594, IMMU-106, TRU-015, AME-133,tositumomab/1-131, tositumomab (Bexxar®), ibritumomab tituxetan(Zevalin®); anti-CD80 antibodies, and etanercept (ENBREL®), as well asanti-T cell antibodies such as anti-CD3 (OKT3), anti-CD4, and the like.

Further, combination therapy with two or more therapeutic agents and ananti-CD20 antibody described herein can also be used for treatment ofdisease states associated with CD20-expressing cells, for example, Bcell-related cancers, and autoimmune and/or inflammatory disorders.Without being limiting, examples include triple combination therapy,where two chemotherapeutic agents are administered in combination withan anti-CD20 antibody described herein, and where a chemotherapeuticagent and another anti-cancer monoclonal antibody (for example,alemtuzumab, rituximab, or anti-CD23 antibody) are administered incombination with an anti-CD20 antibody described herein. Examples ofsuch combinations include, but are not limited to, combinations offludarabine, cyclophosphamide, and the anti-CD20 antibody; andcombinations of fludarabine, another anti-CD20 antibody, for example,rituximab (Rituxan®; IDEC Pharmaceuticals Corp., San Diego, Calif.), andan anti-CD20 antibody of the invention.

A further embodiment of the invention is the use of anti-CD20 antibodiesfor diagnostic monitoring of protein levels in tissue as part of aclinical testing procedure, e.g., to determine the efficacy of a giventreatment regimen. Detection can be facilitated by coupling the antibodyto a detectable substance. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S,or ³H.

VIII. Pharmaceutical Compositions and Administration Methods

Methods of preparing and administering the anti-CD20 antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention to a subject in need thereof are well known to or are readilydetermined by those skilled in the art. The route of administration ofthe anti-CD20 antibody, or antigen-binding fragment, variant, orderivative thereof may be, for example, oral, parenteral, by inhalationor topical. The term parenteral as used herein includes, e.g.,intravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, rectal, or vaginal administration. While all these formsof administration are clearly contemplated as being within the scope ofthe invention, a form for administration would be a solution forinjection, in particular for intravenous or intraarterial injection ordrip. Usually, a suitable pharmaceutical composition for injection maycomprise a buffer (e.g. acetate, phosphate or citrate buffer), asurfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. humanalbumin), etc. However, in other methods compatible with the teachingsherein, anti-CD20 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be delivered directly to thesite of the adverse cellular population thereby increasing the exposureof the diseased tissue to the therapeutic agent.

As previously discussed, anti-CD20 antibodies, or antigen-bindingfragments, variants, or derivatives thereof, of the invention may beadministered in a pharmaceutically effective amount for the in vivotreatment of CD20-expressing cell-mediated diseases such as SLE, PBC,ITP, multiple sclerosis, psoriasis, Crohn's disease, graft rejection,and B-cell lymphoma. In this regard, it will be appreciated that thedisclosed antibodies will be formulated so as to facilitateadministration and promote stability of the active agent. Preferably,pharmaceutical compositions in accordance with the present inventioncomprise a pharmaceutically acceptable, non-toxic, sterile carrier suchas physiological saline, non-toxic buffers, preservatives and the like.For the purposes of the instant application, a pharmaceuticallyeffective amount of an anti-CD20 antibody, or antigen-binding fragment,variant, or derivative thereof, conjugated or unconjugated, shall beheld to mean an amount sufficient to achieve effective binding to atarget and to achieve a benefit, e.g., to ameliorate symptoms of adisease or disorder or to detect a substance or a cell.

The pharmaceutical compositions used in this invention comprisepharmaceutically acceptable carriers, including, e.g., ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol, andwool fat.

Preparations for parenteral administration includes sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. In the subject invention, pharmaceutically acceptable carriersinclude, but are not limited to, 0.01-0.1 M and preferably 0.05 Mphosphate buffer or 0.8% saline. Other common parenteral vehiclesinclude sodium phosphate solutions, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's, or fixed oils. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives may also be present such as for example, antimicrobials,antioxidants, chelating agents, and inert gases and the like.

More particularly, pharmaceutical compositions suitable for injectableuse include sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In such cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It should be stable under the conditions ofmanufacture and storage and will preferably be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Suitableformulations for use in the therapeutic methods disclosed herein aredescribed in Remington's Pharmaceutical Sciences (Mack Publishing Co.)16th ed. (1980).

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

In any case, sterile injectable solutions can be prepared byincorporating an active compound (e.g., an anti-CD20 antibody, orantigen-binding fragment, variant, or derivative thereof, by itself orin combination with other active agents) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedherein, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle, which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying,which yields a powder of an active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The preparations for injections are processed, filled into containerssuch as ampoules, bags, bottles, syringes or vials, and sealed underaseptic conditions according to methods known in the art. Further, thepreparations may be packaged and sold in the form of a kit such as thosedescribed in co-pending U.S. Patent Application No., published as Ser.No. 09/259,337 US-2002-0102208 A1, which is incorporated herein byreference in its entirety. Such articles of manufacture will preferablyhave labels or package inserts indicating that the associatedcompositions are useful for treating a subject suffering from, orpredisposed to a disease or disorder.

Parenteral formulations may be a single bolus dose, an infusion or aloading bolus dose followed with a maintenance dose. These compositionsmay be administered at specific fixed or variable intervals, e.g., oncea day, or on an “as needed” basis.

Certain pharmaceutical compositions used in this invention may be orallyadministered in an acceptable dosage form including, e.g., capsules,tablets, aqueous suspensions or solutions. Certain pharmaceuticalcompositions also may be administered by nasal aerosol or inhalation.Such compositions may be prepared as solutions in saline, employingbenzyl alcohol or other suitable preservatives, absorption promoters toenhance bioavailability, and/or other conventional solubilizing ordispersing agents.

The amount of an anti-CD20 antibody, or fragment, variant, or derivativethereof that may be combined with the carrier materials to produce asingle dosage form will vary depending upon the host treated and theparticular mode of administration. The composition may be administeredas a single dose, multiple doses or over an established period of timein an infusion. Dosage regimens also may be adjusted to provide theoptimum desired response (e.g., a therapeutic or prophylactic response).

In keeping with the scope of the present disclosure, anti-CD20antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention may be administered to a human or other animalin accordance with the aforementioned methods of treatment in an amountsufficient to produce a therapeutic effect. The anti-CD20 antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be administered to such human or other animal in aconventional dosage form prepared by combining the antibody of theinvention with a conventional pharmaceutically acceptable carrier ordiluent according to known techniques. It will be recognized by one ofskill in the art that the form and character of the pharmaceuticallyacceptable carrier or diluent is dictated by the amount of activeingredient with which it is to be combined, the route of administrationand other well-known variables. Those skilled in the art will furtherappreciate that a cocktail comprising one or more species of anti-CD20antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention may prove to be particularly effective.

Effective doses of the compositions of the present invention, fortreatment of CD20-expressing cell-mediated diseases such as SLE, PBC,ITP, multiple sclerosis, psoriasis, Crohn's disease, graft rejection,and B-cell lymphoma vary depending upon many different factors,including means of administration, target site, physiological state ofthe patient, whether the patient is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic. Usually, the patient is a human, but non-human mammalsincluding transgenic mammals can also be treated. Treatment dosages maybe titrated using routine methods known to those of skill in the art tooptimize safety and efficacy.

The amount of at least one anti-CD20 antibody to be administered isreadily determined by one of ordinary skill in the art without undueexperimentation given the disclosure of the present invention. Factorsinfluencing the mode of administration and the respective amount of atleast one anti-CD20 antibody, antigen-binding fragment, variant orderivative thereof include, but are not limited to, the severity of thedisease, the history of the disease, and the age, height, weight,health, and physical condition of the individual undergoing therapy.Similarly, the amount of anti-CD20 antibody, or fragment, variant, orderivative thereof to be administered will be dependent upon the mode ofadministration and whether the subject will undergo a single dose ormultiple doses of this agent. The dose of anti-CD20 antibody, orfragment, or variant, or derivative thereof to be administered is in therange from about 0.0001 to 100 mg/kg, 0.003 mg/kg to about 50 mg/kg, orabout 0.01 mg/kg to about 40 mg/kg. Thus, for example, the dose can be0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 5 mg/kg, 7 mg/kg, 10 mg/kg, 15mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg.

In some embodiments, for treatment of CD20-expressing cell-mediateddiseases such as SLE, PBC, ITP, multiple sclerosis, psoriasis, Crohn'sdisease, graft rejection, and B-cell lymphoma with an anti-CD20antibody, or antigen-binding fragment, variant, or derivative thereof,the dosage can range, e.g., from about 0.0001 to 100 mg/kg, and moreusually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the host body weight. For exampledosages can be 1 mg/kg body weight or 10 mg/kg body weight or within therange of 1-10 mg/kg, preferably at least 1 mg/kg. Doses intermediate inthe above ranges are also intended to be within the scope of theinvention. Subjects can be administered such doses daily, on alternativedays, weekly or according to any other schedule determined by empiricalanalysis. Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kg onconsecutive days, 30 mg/kg on alternate days, or 60 mg/kg weekly. Insome methods, two or more monoclonal antibodies with different bindingspecificities are administered simultaneously, in which case the dosageof each antibody administered falls within the ranges indicated.

Anti-CD20 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be administered on multipleoccasions. Intervals between single dosages can be daily, weekly,monthly or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of target polypeptide or target molecule in thepatient. In some methods, dosage is adjusted to achieve a plasmapolypeptide concentration of 1-1000 μg/ml and in some methods 25-300μg/ml. Alternatively, anti-CD20 antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention can beadministered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency vary dependingon the half-life of the antibody in the patient. The half-life of ananti-CD20 antibody can also be prolonged via fusion to a stablepolypeptide or moeity, e.g., albumin or PEG. In general, humanizedantibodies show the longest half-life, followed by chimeric antibodiesand nonhuman antibodies. In one embodiment, the anti-CD20 antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be administered in unconjugated form, In anotherembodiment, the anti-CD20 antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention can be administeredmultiple times in conjugated form. In still another embodiment,anti-CD20 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be administered in unconjugatedform, then in conjugated form, or vice versa.

In another embodiment of the invention, the method comprisesadministration of multiple doses of anti-CD20 antibody, orantigen-binding fragment, variant, or derivative thereof. The method maycomprise administration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, or more therapeutically effective doses of a pharmaceuticalcomposition comprising an anti-CD20 antibody, fragment, variant, orderivative thereof. The frequency and duration of administration ofmultiple doses of the pharmaceutical compositions comprising theantibody molecule can be readily determined by one of skill in the artwithout undue experimentation given the disclosure herein. Moreover,treatment of a subject with a therapeutically effective amount of anantibody can include a single treatment or, preferably, can include aseries of treatments. In a preferred example, a subject is treated withanti-CD20 antibody, or antigen-binding fragment, variant, or derivativethereof in the range of between about 0.1 to 20 mg/kg body weight, onceper week for between about 1 to 10 weeks, preferably between about 2 to8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. Treatment may occur annually toprevent relapse or upon indication of relapse. It will also beappreciated that the effective dosage of antibody molecule used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

Thus, in one embodiment, the dosing regimen includes a firstadministration of a therapeutically effective dose of at least oneanti-CD20 antibody, or antigen-binding fragment, variant, or derivativethereof, on days 1, 7, 14, and 21 of a treatment period. In anotherembodiment, the dosing regimen includes a first administration of atherapeutically effective dose of at least one anti-CD20 antibody, orantigen-binding fragment, variant, or derivative thereof, on days 1, 2,3, 4, 5, 6, and 7 of a week in a treatment period. Further embodimentsinclude a dosing regimen having a first administration of atherapeutically effective dose of at least one anti-CD20 antibody, orantigen-binding fragment, variant, or derivative thereof, on days 1, 3,5, and 7 of a week in a treatment period; a dosing regimen including afirst administration of a therapeutically effective dose of at least oneanti-CD20 antibody, or antigen-binding fragment, variant, or derivativethereof, on days 1 and 3 of a week in a treatment period; and apreferred dosing regimen including a first administration of atherapeutically effective dose of at least one anti-CD20 antibody, orantigen-binding fragment, variant, or derivative thereof, on day 1 of aweek in a treatment period. The treatment period may comprise 1 week, 2weeks, 3 weeks, a month, 3 months, 6 months, or a year. Treatmentperiods may be subsequent or separated from each other by a day, a week,2 weeks, a month, 3 months, 6 months, or a year.

In some embodiments, the therapeutically effective doses of anti-CD20antibody, or antigen-binding fragment, variant, or derivative thereof,ranges from about 0.0001 mg/kg to about 100 mg/kg, from about 0.003mg/kg to about 50 mg/kg, from about 0.01 mg/kg to about 40 mg/kg, fromabout 0.01 mg/kg to about 30 mg/kg, from about 0.1 mg/kg to about 30mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 1 mg/kg toabout 30 mg/kg, from about 3 mg/kg to about 30 mg/kg, from about 3 mg/kgto about 25 mg/kg, from about 3 mg/kg to about 20 mg/kg, from about 5mg/kg to about 15 mg/kg, or from about 7 mg/kg to about 12 mg/kg. Thus,for example, the dose of any one anti-CD20 antibody, or antigen-bindingfragment, variant, or derivative thereof, can be 0.003 mg/kg, 0.01mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg,2 mg/kg, 2.5 mg/kg, 3 mg/kg, 5 mg/kg, 7 mg/kg, 10 mg/kg, 15 mg/kg, 20mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, orother such doses falling within the range of about 0.0001 mg/kg to about100 mg/kg. The same therapeutically effective dose of an anti-CD20antibody, or antigen-binding fragment, variant, or derivative thereof,can be administered throughout each week of antibody dosing.Alternatively, different therapeutically effective doses of an anti-CD20antibody, or antigen-binding fragment, variant, or derivative thereof,can be used over the course of a treatment period.

IX. Use of Anti-CD20 Antibodies in the Manufacture of Medicaments

The present invention also provides for the use of an anti-CD20 antibodyor antigen-binding fragment, variant, or derivative thereof in themanufacture of a medicament for treating a subject for a cancercharacterized by neoplastic B cell growth, wherein the medicament iscoordinated with treatment with at least one other cancer therapy.Cancers characterized by neoplastic B cell growth include, but are notlimited to, the B cell-related cancers discussed herein above, forexample, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, multiplemyeloma, B cell lymphoma, high-grade B cell lymphoma, intermediate-gradeB cell lymphoma, low-grade B cell lymphoma, B cell acute lymphoblasticleukemia, myeloblastic leukemia, Hodgkin's disease, plasmacytoma,follicular lymphoma, follicular small cleaved lymphoma, follicular largecell lymphoma, follicular mixed small cleaved lymphoma, diffuse smallcleaved cell lymphoma, diffuse small lymphocytic lymphoma,prolymphocytic leukemia, lymphoplamacytic lymphoma, marginal zonelymphoma, mucosal associated lymphoid tissue lymphoma, monocytoid B celllymphoma, splenic lymphoma, hairy cell leukemia, diffuse large celllymphoma, mediastinal large B cell lymphoma, lymphomatoidgranulomatosis, intravascular lymphomatosis, diffuse mixed celllymphoma, diffuse large cell lymphoma, immunoblastic lymphoma, Burkitt'slymphoma, AIDS-related lymphoma, and mantle cell lymphoma.

By “coordinated” is intended the medicament comprising the anti-CD20antibody or antigen-binding fragment, variant, or derivative thereof isto be used either prior to, during, or after treatment of the subjectwith at least one other cancer therapy. Examples of other cancertherapies include, but are not limited to, surgery; radiation therapy;chemotherapy, optionally in combination with autologous bone marrowtransplant, where suitable chemotherapeutic agents include, but are notlimited to, fludarabine or fludarabine phosphate, chlorambucil,vincristine, pentostatin, 2-chlorodeoxyadenosine (cladribine),cyclophosphamide, doxorubicin, prednisone, and combinations thereof, forexample, anthracycline-containing regimens such as CAP(cyclophosphamide, doxorubicin plus prednisone), CHOP (cyclophosphamide,vincristine, prednisone plus doxorubicin), VAD (vincritsine,doxorubicin, plus dexamethasone), MP (melphalan plus prednisone), andother cytotoxic and/or therapeutic agents used in chemotherapy such asmitoxantrone, daunorubicin, idarubicin, asparaginase, andantimetabolites, including, but not limited to, cytarabine,methotrexate, 5-fluorouracil decarbazine, 6-thioguanine,6-mercaptopurine, and nelarabine; other anti-cancer monoclonal antibodytherapy (for example, alemtuzumab (Campath®) or other anti-CD52 antibodytargeting the CD52 cell-surface glycoprotein on malignant B cells;rituximab (Rituxan®), the fully human antibody HuMax-CD20, R-1594,IMMU-106, TRU-015, AME-133, tositumomab/1-131 tositumomab (Bexxar®),ibritumomab tiuxetan (Zevalin®), or any other therapeutic anti-CD20antibody targeting the CD20 antigen on malignant B cells; anti-CD 19antibody (for example, MT103, a bispecific antibody); anti-CD22 antibody(for example, the humanized monoclonal antibody epratuzumab);bevacizumab (Avastin®) or other anti-cancer antibody targeting humanvascular endothelial growth factor; anti-CD22 antibody targeting theCD22 antigen on malignant B cells (for example, the monoclonal antibodyBL-22, an alphaCD22 toxin); α-M-CSF antibody targeting macrophage colonystimulating factor; antibodies targeting the receptor activator ofnuclear factor-kappaB (RANK) and its ligand (RANKL), which areoverexpressed in multiple myeloma; anti-CD23 antibody targeting the CD23antigen on malignant B cells (for example, IDEC-152); anti-CD38 antibodytargeting the CD38 antigen on malignant B cells; antibodies targetingmajor histocompatibility complex class II receptors (anti-MHCantibodies) expressed on malignant B cells; anti-CD40 antibodies (forexample, SGN-40) targeting the CD40 antigen on malignant B cells; andantibodies targeting tumor necrosis factor-related apoptosis-inducingligand receptor 1 (TRAIL-R1) (for example, the agonistic humanmonoclonal antibody HGS-ETR1) expressed on a number of solid tumors andtumors of hematopoietic origin); small molecule-based cancer therapy,including, but not limited to, microtubule and/or topoisomeraseinhibitors (for example, the mitotic inhibitor dolastatin and dolastatinanalogues; the tubulin-binding agent T900607; XL119; and thetopoisomerase inhibitor aminocamptothecin), SDX-105 (bendamustinehydrochloride), ixabepilone (an epothilone analog, also referred to asBMS-247550), protein kinase C inhibitors, for example, midostaurin((PKC-412, CGP 41251, N-benzoylstaurosporine), pixantrone, eloxatin (anantineoplastic agent), ganite (gallium nitrate), Thalomid®(thalidomide), immunomodulatory derivatives of thalidomide (for example,revlimid (formerly revimid)), Affinitak™ (antisense inhibitor of proteinkinase C-alpha), SDX-101 (R-etodolac, inducing apoptosis of malignantlymphocytes), second-generation purine nucleoside analogs such asclofarabine, inhibitors of production of the protein Bcl-2 by cancercells (for example, the antisense agents oblimersen and Genasense®),proteasome inhibitors (for example, Velcade™ (bortezomib)), smallmolecule kinase inhibitors (for example, CHIR-258), small molecule VEGFinhibitors (for example, ZD-6474), small molecule inhibitors of heatshock protein (HSP) 90 (for example, 17-AAG), small molecule inhibitorsof histone deacetylases (for example, hybrid/polar cytodifferentiationHPC) agents such as suberanilohydroxamic acid (SAHA), and FR-901228) andapoptotic agents such as Trisenox® (arsenic trioxide) and Xcytrin®(motexafin gadolinium); vaccine/immunotherapy-based cancer therapies,including, but not limited to, vaccine approaches (for example, Id-KLH,oncophage, vitalethine), personalized immunotherapy or active idiotypeimmunotherapy (for example, MyVax® Personalized Immunotherapy, formallydesignated GTOP-99), Promune® (CpG 7909, a synthetic agonist fortoll-like receptor 9 (TLR9)), interferon-alpha therapy, interleukin-2(IL-2) therapy, IL-12 therapy; IL-15 therapy, and IL-21 therapy; steroidtherapy; or other cancer therapy; where treatment with the additionalcancer therapy, or additional cancer therapies, occurs prior to, during,or subsequent to treatment of the subject with the medicament comprisingthe anti-CD20 antibody or antigen-binding fragment, variant, orderivative thereof, as noted herein above.

In some embodiments, the present invention provides for the use of theanti-CD20 antibody or antigen-binding fragment, variant, or derivativethereof in the manufacture of a medicament for treating a B celllymphoma, for example non-Hodgkin's lymphoma, in a subject, wherein themedicament is coordinated with treatment with at least one other cancertherapy selected from the group consisting of chemotherapy, anti-cancerantibody therapy, small molecule-based cancer therapy, andvaccine/immunotherapy-based cancer therapy, wherein the medicament is tobe used either prior to, during, or after treatment of the subject withthe other cancer therapy or, in the case of multiple combinationtherapies, either prior to, during, or after treatment of the subjectwith the other cancer therapies.

Thus, for example, in some embodiments, the invention provides for theuse of the monoclonal antibody 1589 or other anti-CD20 antibodies of theinvention, or antigen-binding fragment, variant, or derivative thereof,in the manufacture of a medicament for treating a B cell lymphoma, forexample, non-Hodgkin's lymphoma, in a subject, wherein the medicament iscoordinated with treatment with chemotherapy, where the chemotherapeuticagent is selected from the group consisting of cytoxan, doxorubicin,vincristine, prednisone, and combinations thereof, for example CHOP. Inother embodiments, the invention provides for the use of the monoclonalantibody 1589, or antigen-binding fragment thereof, in the manufactureof a medicament for treating a B cell lymphoma, for examplenon-Hodgkin's lymphoma, in a subject, wherein the medicament iscoordinated with treatment with at least one other anti-cancer antibodyselected from the group consisting of alemtuzumab (Campath®) or otheranti-CD52 antibody targeting the CD52 cell-surface glycoprotein onmalignant B cells; rituximab (Rituxan®), the fully human antibodyHuMax-CD20, R-1594, IMMU-106, TRU-015, AME-133, tositumomab/I-131tositumomab (Bexxar®), ibritumomab tiuxetan (Zevalin®), or any othertherapeutic anti-CD20 antibody targeting the CD20 antigen on malignant Bcells; anti-CD19 antibody (for example, MT103, a bispecific antibody);anti-CD22 antibody (for example, the humanized monoclonal antibodyepratuzumab); bevacizumab (Avastin®) or other anti-cancer antibodytargeting human vascular endothelial growth factor; and any combinationsthereof; wherein the medicament is to be used either prior to, during,or after treatment of the subject with the other cancer therapy or, inthe case of multiple combination therapies, either prior to, during, orafter treatment of the subject with the other cancer therapies.

In yet other embodiments, the present invention provides for the use ofthe monoclonal antibody 1589, or antigen-binding fragment, variant, orderivative thereof, in the manufacture of a medicament for treating a Bcell lymphoma, for example non-Hodgkin's lymphoma, in a subject, whereinthe medicament is coordinated with treatment with at least one othersmall molecule-based cancer therapy selected from the group consistingof microtubule and/or topoisomerase inhibitors (for example, the mitoticinhibitor dolastatin and dolastatin analogues; the tubulin-binding agentT900607; XL119; and the topoisomerase inhibitor aminocamptothecin),SDX-105 (bendamustine hydrochloride), ixabepilone (an epothilone analog,also referred to as BMS-247550), protein kinase C inhibitors, forexample, midostaurin ((PKC-412, CGP 41251, N-benzoylstaurosporine),pixantrone, eloxatin (an antineoplastic agent), ganite (galliumnitrate), Thalomid® (thalidomide), an apoptotic agent such as Xcytrin®(motexafin gadolinium), inhibitors of production of the protein Bcl-2 bycancer cells (for example, the antisense agents oblimersen andGenasense®), nelarabine, and any combinations thereof; wherein themedicament is to be used either prior to, during, or after treatment ofthe subject with the other cancer therapy or, in the case of multiplecombination therapies, either prior to, during, or after treatment ofthe subject with the other cancer therapies.

In still other embodiments, the present invention provides for the useof the monoclonal antibody 1589, or antigen-binding fragment, variant,or derivative thereof, in the manufacture of a medicament for treating aB cell lymphoma, for example non-Hodgkin's lymphoma, in a subject,wherein the medicament is coordinated with treatment with at least oneother vaccine/immunotherapy-based cancer therapy selected from the groupconsisting of vaccine approaches (for example, Id-KLH, oncophage,vitalethine), personalized immunotherapy or active idiotypeimmunotherapy (for example, MyVax® Personalized Immunotherapy, formallydesignated GTOP-99), Promune® (CpG 7909, a synthetic agonist fortoll-like receptor 9 (TLR9)), interleukin-2 (IL-2) therapy, IL-12therapy; IL-15 therapy, and IL-21 therapy, and any combinations thereof;wherein the medicament is to be used either prior to, during, or aftertreatment of the subject with the other cancer therapy or, in the caseof multiple combination therapies, either prior to, during, or aftertreatment of the subject with the other cancer therapies.

In some embodiments, the present invention provides for the use of theanti-CD20 antibody, for example, the monoclonal antibody 1589, orantigen-binding fragment, variant, or derivative thereof, in themanufacture of a medicament for treating a B cell-related leukemia, forexample B-cell acute lymphocytic leukemia (B-ALL), in a subject, whereinthe medicament is coordinated with treatment with at least one othercancer therapy selected from the group consisting of chemotherapy andanti-metabolite therapy, wherein the medicament is to be used eitherprior to, during, or after treatment of the subject with the othercancer therapy or, in the case of multiple combination therapies, eitherprior to, during, or after treatment of the subject with the othercancer therapies. Examples of such embodiments include, but are notlimited to, those instances where the medicament comprising theanti-CD20 antibody, for example, the monoclonal antibody 1589, orantigen-binding fragment, variant, or derivative thereof, is coordinatedwith treatment with a chemotherapeutic agent or anti-metabolite selectedfrom the group consisting of cytoxan, doxorubicin, vincristine,prednisone, cytarabine, mitoxantrone, idarubicin, asparaginase,methotrexate, 6-thioguanine, 6-mercaptopurine, and combinations thereof;wherein the medicament is to be used either prior to, during, or aftertreatment of the subject with the other cancer therapy or, in the caseof multiple combination therapies, either prior to, during, or aftertreatment of the subject with the other cancer therapies. In one suchexample, the medicament is coordinated with treatment with cytarabineplus daunorubicin, cytarabine plus mitoxantrone, and/or cytarabine plusidarubicin; wherein the medicament is to be used either prior to,during, or after treatment of the B-ALL subject with the other cancertherapy or, in the case of multiple combination therapies, either priorto, during, or after treatment of the subject with the other cancertherapies.

The invention also provides for the use of an anti-CD20 antibody, forexample, the monoclonal antibody 1589 disclosed herein, orantigen-binding fragment, variant, or derivative thereof, in themanufacture of a medicament for treating a subject for a cancercharacterized by neoplastic B cell growth, including the B cell-relatedcancers described herein above, wherein the medicament is used in asubject that has been pretreated with at least one other cancer therapy.By “pretreated” or “pretreatment” is intended the subject has receivedone or more other cancer therapies (i.e., been treated with at least oneother cancer therapy) prior to receiving the medicament comprising theanti-CD20 antibody or antigen-binding fragment, variant, or derivativethereof. “Pretreated” or “pretreatment” includes subjects that have beentreated with at least one other cancer therapy within 2 years, within 18months, within 1 year, within 6 months, within 2 months, within 6 weeks,within 1 month, within 4 weeks, within 3 weeks, within 2 weeks, within 1week, within 6 days, within 5 days, within 4 days, within 3 days, within2 days, or even within 1 day prior to initiation of treatment with themedicament comprising the anti-CD20 antibody, for example, themonoclonal antibody 1589 disclosed herein, or antigen-binding fragment,variant, or derivative thereof. It is not necessary that the subject wasa responder to pretreatment with the prior cancer therapy, or priorcancer therapies. Thus, the subject that receives the medicamentcomprising the anti-CD20 antibody or antigen-binding fragment, variant,or derivative thereof could have responded, or could have failed torespond (i.e., the cancer was refractory), to pretreatment with theprior cancer therapy, or to one or more of the prior cancer therapieswhere pretreatment comprised multiple cancer therapies. Examples ofother cancer therapies for which a subject can have receivedpretreatment prior to receiving the medicament comprising the anti-CD20antibody or antigen-binding fragment, variant, or derivative thereofinclude, but are not limited to, surgery; radiation therapy;chemotherapy, optionally in combination with autologous bone marrowtransplant, where suitable chemotherapeutic agents include, but are notlimited to, those listed herein above; other anti-cancer monoclonalantibody therapy, including, but not limited to, those anti-cancerantibodies listed herein above; small molecule-based cancer therapy,including, but not limited to, the small molecules listed herein above;vaccine/immunotherapy-based cancer therapies, including, but limited to,those listed herein above; steroid therapy; other cancer therapy; or anycombination thereof.

“Treatment” in the context of coordinated use of a medicament describedherein with one or more other cancer therapies is herein defined as theapplication or administration of the medicament or of the other cancertherapy to a subject, or application or administration of the medicamentor other cancer therapy to an isolated tissue or cell line from asubject, where the subject has a cancer characterized by neoplastic Bcell growth, a symptom associated with such a cancer, or apredisposition toward development of such a cancer, where the purpose isto cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve,or affect the cancer, any associated symptoms of the cancer, or thepredisposition toward the development of the cancer.

The present invention also provides for the use of an anti-CD20 antibodyor antigen-binding fragment, variant, or derivative thereof in themanufacture of a medicament for treating an autoimmune disease and/orinflammatory disease in a subject, wherein the medicament is coordinatedwith treatment with at least one other therapy. By “coordinated” isintended the medicament is to be used either prior to, during, or aftertreatment of the subject with at least one other therapy for theautoimmune disease and/or inflammatory disease. Examples of othertherapies include, but are not limited to, those described herein above,i.e., surgery or surgical procedures (e.g. splenectomy, lymphadenectomy,thyroidectomy, plasmaphoresis, leukophoresis, cell, tissue, or organtransplantation, organ perfusion, intestinal procedures, and the like),radiation therapy, therapy such as steroid therapy and non-steroidaltherapy, hormone therapy, cytokine therapy, therapy with dermatologicalagents (for example, topical agents used to treat skin conditions suchas allergies, contact dermatitis, and psoriasis), immunosuppressivetherapy, and other anti-inflammatory monoclonal antibody therapy, andthe like, where treatment with the additional therapy, or additionaltherapies, occurs prior to, during, or subsequent to treatment of thesubject with the medicament comprising the anti-CD20 antibody orantigen-binding fragment, variant, or derivative thereof, as notedherein above. In one such embodiment, the present invention provides forthe use of the monoclonal antibody 1589, or antigen-binding fragment,variant, or derivative thereof, in the manufacture of a medicament fortreating an autoimmune disease and/or inflammatory disease in a subject,wherein the medicament is coordinated with treatment with at least oneother therapy as noted herein above.

In some embodiments, the medicament comprising the anti-CD20 antibody,for example, the monoclonal antibody 1589 disclosed herein, orantigen-binding fragment, variant, or derivative thereof, is coordinatedwith treatment with two other therapies. Where the medicament comprisingthe anti-CD20 antibody is coordinated with two other therapies, use ofthe medicament can be prior to, during, or after treatment of thesubject with either or both of the other therapies.

The invention also provides for the use of an anti-CD20 antibody, forexample, the monoclonal antibody 1589 disclosed herein, orantigen-binding fragment, variant, or derivative thereof, in themanufacture of a medicament for treating an autoimmune disease and/orinflammatory disease in a subject, wherein the medicament is used in asubject that has been pretreated with at least one other therapy. By“pretreated” or “pretreatment” is intended the subject has been treatedwith one or more other therapies prior to receiving the medicamentcomprising the anti-CD20 antibody or antigen-binding fragment, variant,or derivative thereof. “Pretreated” or “pretreatment” includes subjectsthat have been treated with the other therapy, or other therapies,within 2 years, within 18 months, within 1 year, within 6 months, within2 months, within 6 weeks, within 1 month, within 4 weeks, within 3weeks, within 2 weeks, within 1 week, within 6 days, within 5 days,within 4 days, within 3 days, within 2 days, or even within 1 day priorto initiation of treatment with the medicament comprising the anti-CD20antibody, for example, the monoclonal antibody 1589 disclosed herein, orantigen-binding fragment, variant, or derivative thereof. It is notnecessary that the subject was a responder to pretreatment with theprior therapy, or prior therapies. Thus, the subject that receives themedicament comprising the anti-CD20 antibody or antigen-bindingfragment, variant, or derivative thereof could have responded, or couldhave failed to respond, to pretreatment with the prior therapy, or toone or more of the prior therapies where pretreatment comprised multipletherapies.

“Treatment” in the context of coordinated use of a medicament describedherein with one or more other autoimmune disease and/or inflammatorydisease therapies is herein defined as the application or administrationof the medicament or of the other therapy to a subject, or applicationor administration of the medicament or other therapy to an isolatedtissue or cell line from a subject, where the subject has an autoimmunedisease and/or inflammatory disease associated with CD20-expressingcells, a symptom associated with such a disease, or a predispositiontoward development of such a disease, where the purpose is to cure,heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affectthe disease, any associated symptoms of the disease, or thepredisposition toward the development of the disease.

X. Diagnostics

The invention further provides a diagnostic method useful duringdiagnosis of CD20-expressing cell-mediated diseases such as SLE, PBC,ITP, multiple sclerosis, psoriasis, Crohn's disease, graft rejection,and B-cell lymphoma, which involves measuring the expression level ofCD20 protein or transcript in tissue or other cells or body fluid froman individual and comparing the measured expression level with astandard CD20 expression level in normal tissue or body fluid, wherebyan increase in the expression level compared to the standard isindicative of a disorder.

The anti-CD20 antibodies of the invention and antigen-binding fragments,variants, and derivatives thereof, can be used to assay CD20 proteinlevels in a biological sample using classical immunohistological methodsknown to those of skill in the art (e.g., see Jalkanen, et al. (1985) J.Cell. Biol. 101:976-985; Jalkanen et al. (1987) J. Cell Biol.105:3087-3096). Other antibody-based methods useful for detecting CD20protein expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA), immunoprecipitation, or western blotting.Suitable assays are described in more detail elsewhere herein.

By “assaying the expression level of CD20 polypeptide” is intendedqualitatively or quantitatively measuring or estimating the level ofCD20 polypeptide in a first biological sample either directly (e.g., bydetermining or estimating absolute protein level) or relatively (e.g.,by comparing to the disease associated polypeptide level in a secondbiological sample). Preferably, CD20 polypeptide expression level in thefirst biological sample is measured or estimated and compared to astandard CD20 polypeptide level, the standard being taken from a secondbiological sample obtained from an individual not having the disorder orbeing determined by averaging levels from a population of individualsnot having the disorder. As will be appreciated in the art, once the“standard” CD20 polypeptide level is known, it can be used repeatedly asa standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, cell line, tissue culture, or other source of cellspotentially expressing CD20. Methods for obtaining tissue biopsies andbody fluids from mammals are well known in the art.

XI. Immunoassays

Anti-CD20 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays that can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al., eds,(1994) Current Protocols in Molecular Biology (John Wiley & Sons, Inc.,NY) Vol. 1, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 MNaCl, 0.01 M sodium phosphate atpH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal., eds, (1994) Current Protocols in Molecular Biology (John Wiley &Sons, Inc., NY) Vol. 1 at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), incubating the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, incubating the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 1251) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al., eds, (1994) Current Protocols in Molecular Biology (John Wiley &Sons, Inc., NY) Vol. 1 at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96-wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al., eds, (1994) Current Protocols in Molecular Biology (JohnWiley & Sons, Inc., NY) Vol. 1 at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated with theantibody of interest conjugated to a labeled compound (e.g., ³H or ¹²⁵I)in the presence of increasing amounts of an unlabeled second antibody.

Anti-CD20 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention, additionally, can be employedhistologically, as in immunofluorescence, immunoelectron microscopy ornon-immunological assays, for in situ detection of cancer antigen geneproducts or conserved variants or peptide fragments thereof. In situdetection may be accomplished by removing a histological specimen from apatient, and applying thereto a labeled anti-CD20 antibody, orantigen-binding fragment, variant, or derivative thereof, preferablyapplied by overlaying the labeled antibody (or fragment) onto abiological sample. Through the use of such a procedure, it is possibleto determine not only the presence of CD20 protein, or conservedvariants or peptide fragments, but also its distribution in the examinedtissue. Using the present invention, those of ordinary skill willreadily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

Immunoassays and non-immunoassays for CD20 gene products or conservedvariants or peptide fragments thereof will typically comprise incubatinga sample, such as a biological fluid, a tissue extract, freshlyharvested cells, or lysates of cells which have been incubated in cellculture, in the presence of a detectably labeled antibody capable ofbinding to CD20 or conserved variants or peptide fragments thereof, anddetecting the bound antibody by any of a number of techniques well knownin the art.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled anti-CD20 antibody, orantigen-binding fragment, variant, or derivative thereof. The solidphase support may then be washed with the buffer a second time to removeunbound antibody. Optionally the antibody is subsequently labeled. Theamount of bound label on solid support may then be detected byconventional means.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-CD20 antibody, orantigen-binding fragment, variant, or derivative thereof may bedetermined according to well known methods. Those skilled in the artwill be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation.

There are a variety of methods available for measuring the affinity ofan antibody-antigen interaction, but relatively few for determining rateconstants. Most of the methods rely on either labeling antibody orantigen, which inevitably complicates routine measurements andintroduces uncertainties in the measured quantities.

Surface plasmon reasonance (SPR) as performed on BIAcore offers a numberof advantages over conventional methods of measuring the affinity ofantibody-antigen interactions including: (i) no requirement to labeleither antibody or antigen; (ii) antibodies do not need to be purifiedin advance, cell culture supernatant can be used directly; (iii)real-time measurements, allowing rapid semi-quantitative comparison ofdifferent monoclonal antibody interactions, are enabled and aresufficient for many evaluation purposes; (iv) biospecific surface can beregenerated so that a series of different monoclonal antibodies caneasily be compared under identical conditions; (v) analytical proceduresare fully automated, and extensive series of measurements can beperformed without user intervention. BIAapplications Handbook, versionAB (reprinted 1998), BIACORE code No. BR-1001-86; BIAtechnologyHandbook, version AB (reprinted 1998), BIACORE code No. BR-1001-84.

SPR based binding studies require that one member of a binding pair beimmobilized on a sensor surface. The binding partner immobilized isreferred to as the ligand. The binding partner in solution is referredto as the analyte. In some cases, the ligand is attached indirectly tothe surface through binding to another immobilized molecule, which isreferred as the capturing molecule. SPR response reflects a change inmass concentration at the detector surface as analytes bind ordissociate.

Based on SPR, real-time BIAcore measurements monitor interactionsdirectly as they happen. The technique is well suited to determinationof kinetic parameters. Comparative affinity ranking is extremely simpleto perform, and both kinetic and affinity constants can be derived fromthe sensorgram data.

When analyte is injected in a discrete pulse across a ligand surface,the resulting sensorgram can be divided into three essential phases: (i)Association of analyte with ligand during sample injection; (ii)Equilibrium or steady state during sample injection, where the rate ofanalyte binding is balanced by dissociation from the complex; (iii)

Dissociation of analyte from the surface during buffer flow.

The association and dissociation phases provide information on thekinetics of analyte-ligand interaction (k_(a) and k_(d), the rates ofcomplex formation and dissociation, k_(d)/k_(a)=K_(D)). The equilibriumphase provides information on the affinity of the analyte-ligandinteraction (K_(D)).

BIAevaluation software provides comprehensive facilities for curvefitting using both numerical integration and global fitting algorithms.With suitable analysis of the data, separate rate and affinity constantsfor interaction can be obtained from simple BIAcore investigations. Therange of affinities measurable by this technique is very broad, rangingfrom mM to pM.

Epitope specificity is an important characteristic of a monoclonalantibody. Epitope mapping with BIAcore, in contrast to conventionaltechniques using radioimmunoassay, ELISA or other surface adsorptionmethods, does not require labeling or purified antibodies, and allowsmulti-site specificity tests using a sequence of several monoclonalantibodies. Additionally, large numbers of analyses can be processedautomatically.

Pair-wise binding experiments test the ability of two MAbs to bindsimultaneously to the same antigen. MAbs directed against separateepitopes will bind independently, whereas MAbs directed againstidentical or closely related epitopes will interfere with each other'sbinding. These binding experiments with BIAcore are straightforward tocarry out.

For example, one can use a capture molecule to bind the first Mab,followed by addition of antigen and second MAb sequentially. Thesensorgrams will reveal: 1. how much of the antigen binds to first Mab,2. to what extent the second MAb binds to the surface-attached antigen,3. if the second MAb does not bind, whether reversing the order of thepair-wise test alters the results.

Peptide inhibition is another technique used for epitope mapping. Thismethod can complement pair-wise antibody binding studies, and can relatefunctional epitopes to structural features when the primary sequence ofthe antigen is known. Peptides or antigen fragments are tested forinhibition of binding of different MAbs to immobilized antigen. Peptideswhich interfere with binding of a given MAb are assumed to bestructurally related to the epitope defined by that MAb.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, Sambrook etal., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; ColdSpring Harbor Laboratory Press); Sambrook et al., ed. (1992) MolecularCloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D.N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984)Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hamesand Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins,eds. (1984) Transcription And Translation; Freshney (1987) Culture OfAnimal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRLPress) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; thetreatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller andCalos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (ColdSpring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols.154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods InCell And Molecular Biology (Academic Press, London); Weir and Blackwell,eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV;Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al. (1989) CurrentProtocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

General principles of antibody engineering are set forth in Borrebaeck,ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). Generalprinciples of protein engineering are set forth in Rickwood et al., eds.(1995) Protein Engineering, A Practical Approach (IRL Press at OxfordUniv. Press, Oxford, Eng.). General principles of antibodies andantibody-hapten binding are set forth in: Nisonoff (1984) MolecularImmunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward(1984) Antibodies, Their Structure and Function (Chapman and Hall, NewYork, N.Y.). Additionally, standard methods in immunology known in theart and not specifically described are generally followed as in CurrentProtocols in Immunology, John Wiley & Sons, New York; Stites et al.,eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange,Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods inCellular Immunology (W.H. Freeman and Co., NY).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination(John Wiley & Sons, NY); Kennett et al., eds. (1980) MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses (PlenumPress, NY); Campbell (1984) “Monoclonal Antibody Technology” inLaboratory Techniques in Biochemistry and Molecular Biology, ed. Burdenet al., (Elsevere, Amsterdam); Goldsby et al., eds. (2000) KubyImmunology (4th ed.; H. Freemand & Co.); Roitt et al. (2001) Immunology(6th ed.; London: Mosby); Abbas et al. (2005) Cellular and MolecularImmunology (5th ed.; Elsevier Health Sciences Division); Kontermann andDubel (2001) Antibody Engineering (Springer Verlan); Sambrook andRussell (2001) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Press); Lewin (2003) Genes VIII (Prentice Hall2003); Harlow andLane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press);Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Optimization of Humanized Murine Anti-CD20Antibody

A humanized murine anti-CD20 monoclonal antibody, mAb 1097, wasengineered within one or more of the complementarity determining regions(CDRs) of its variable heavy (V_(H); see SEQ ID NO:29) and variablelight (V_(K); see SEQ ID NO:10) domains to yield optimized mAbs havingincreased CDC function. The initial humanized murine anti-CD20 mAb is ahumanized form of the chimeric murine/human anti-CD20 antibody known asrituximab (i.e., mAb C2B8; see U.S. Pat. No. 5,736,137). The three CDRsof the original V_(H) domain (designated H1286) and original V_(K)domain (designated L373) within the humanized murine anti-CD20 mAb areshown in FIG. 1. The CDR2 of L373 (shown in SEQ ID NO:9) was derivedfrom a human V_(K) and not a murine V_(K).

Initial optimization steps included modifications to the starting CDR3(designated as “271” in FIG. 1; SEQ ID NO:30) of the H1286 V_(H) domain(see FIG. 2). The 271 CDR3 sequence is equivalent to that for the CDR3of the variable domain within the heavy chain of mAb C2B8. The initialmodifications to 271 CDR3 included:

1) a tyrosine to asparagine (Y→N) substitution at position 9 of thestarting CDR3 and a valine to asparagine (V→N) substitution at position12 of the starting CDR3 to yield the “1236” optimized CDR3 (SEQ IDNO:1);

2) a glycine to alanine (G→A) substitution at position 5 of the initialCDR3 in addition to the Y→N and V→N substitutions at positions 9 and 12,respectively, to yield the “1237” optimized CDR3 (SEQ ID NO:2); or

3) a valine to aspartic acid (V→D) substitution at position 12 of theinitial CDR3 in addition to the G→A and Y→N substitutions at positions 5and 9, respectively, to yield the “1238” optimized CDR3 (SEQ ID NO:3).

The individual coding sequences for the optimized CDR3s are set forth inSEQ ID NO:24 (encoding 1236 optimized CDR3), SEQ ID NO:25 (encoding 1237optimized CDR3), and SEQ ID NO:26 (encoding 1238 optimized CDR3).

These optimized CDR3s were then engineered into the original humanizedV_(H) domain framework of H1286 to yield the following optimized V_(H)domains: H1569 (SEQ ID NO:12), comprising the 1236 optimized CDR3 of SEQID NO:1; H1570 (SEQ ID NO:13), comprising the 1237 optimized CDR3 of SEQID NO:2; and H1571 (SEQ ID NO:14), comprising the 1238 optimized CDR3 ofSEQ ID NO:3) (see FIG. 3). The coding sequences for these optimizedV_(H) domains are shown in FIG. 4, and set forth in SEQ ID NO:19(encoding H1569), SEQ ID NO:20 (encoding H1570), and SEQ ID NO:21(encoding H1571). Each of these optimized V_(H) domains was then pairedwith the original (L373) V_(K) domain (see FIG. 5; SEQ ID NO:10; codingsequence set forth in SEQ ID NO:23) to yield the following optimizedhumanized mABs: mAb 1236, mAb 1237, and mAb 1238.

Example 2 Binding and Functional Characteristics of Optimized HumanizedMurine Anti-CD20 Monoclonal Antibodies

The optimized humanized mAb 1236, mAb 1237, and mAb 1238 described inExample 1 were assessed for their respective binding and functionalcharacteristics. For each assay, mAb 271, having the identical sequenceto rituximab (IDEC-C2B8; Rituxan®; IDEC Pharmaceuticals Corp., SanDiego, Calif.), served as the positive control.

Binding specificity was assessed by Fluorometric Microvolume AssayTechnology (FMAT, Applied Biosystems 8200 Cellular Detection System) andFlow Cytometry, which tabulates Mean Fluorescence Intensities (MFI) ofstaining on CD20 positive (CD20⁺) and CD20 negative cell lines. FMATanalysis demonstrated binding specificity for CD20 (FIGS. 6A-6D). Flowcytometry analysis demonstrated binding of these optimized humanizedanti-CD20 mAbs to CD20+ Daudi and EB1 cells (Table 2). TABLE 2 Bindingto CD20 membrane positive and negative cell lines Flow Cytometry Results(Mean Fluorescence Intensities). Daudi EB1 K562 U266 CD20 CD20 CD20 CD20MAb Positive Positive Negative Negative  271 145 429 10 9 1236 145 40912 11 1237 132 395 14 11 1238 114 389 11 10  11 8 8 6 5 (Negativecontrol)

In order to assess the epitope recognized by mAb1236, mAb 1237, and mAb1238, cells and CD20-specific mouse 2H7 antibody (or mouse ISO) @3 μg/mlwere added to the FMAT plate followed by incubation at room temperaturefor 2 hours. The test mAb (30 ng/ml) and an anti-human IgFc-Alexa 647were then added, and fluorescence signal detected by FMAT. A decrease insignal with mouse 2H7 (m2H7) indicates epitope blocking.

As can be seen in FIG. 7, and as expected, mAb 1236, mAb 1237, and mAb1238 all recognize the same epitope as mAb 271 (rituximab-sequenceantibody).

Complement dependent cytotoxicity (CDC) and antibody-dependent cellularcytotoxicity (ADCC) were assessed. For CDC, an antibody off-rate assaywas used. In this manner, target CD20⁺Daudi cells were loaded with ⁵¹Cr,200 μCi for 2 hours, and then washed. The washed Daudi cells wereincubated with 10 μg/ml of respective mAb for 15 minutes at 25° C.Target Daudi cells were then washed thoroughly and incubated at 37° C.Following 0, 1, 2, 4, and 6 hour incubations at 37° C., the cells wereincubated with 6% human serum (complement source) for 45 minutes.Controls included spontaneous and maximum ⁵¹Cr release, mAb with Daudicells alone, and serum with Daudi cells alone at all concentrations.Supernatents were then processed and released ⁵¹Cr counted via gammacounter. Antibody-specific CDC was determined by subtracting out mAb andserum alone contributions to lysis.

As can be seen in FIG. 8, mAb 1236, mAb 1237, and mAb 1238 havesignificantly better CDC functional activity than mAb 271(rituximab-sequence antibody).

ADCC activity was assayed by loading target Daudi cells with ⁵¹Cr, 200μCi for 2 hours, and following with a wash. The washed Daudi cells wereincubated with titrated concentrations of respective mAbs and freshlyprepared PBMCs (NK source) at 50:1 for 4 hours. Controls includedspontaneous and maximum ⁵¹Cr release, mAb with Daudi cells alone, andPBMC with Daudi cells alone at all concentrations. Supernatents werethen processed and released ⁵¹Cr counted via gamma counter.Antibody-specific ADCC was determined by subtracting out PBMCcontribution to lysis.

As can be seen in FIG. 9, ADCC activity increased with increasing mAbconcentration for all mAbs tested. At all mAb concentrations, the mAb1236, mAb 1237, and mAb1238 have ADCC activity that is equivalent tothat observed for the control mAb 271 (rituximab-sequence antibody).

Apoptosis activity was also assessed using a direct apoptosis assay thatis CDC and ADCC independent. In this manner, Ramos cells (NHL) wereincubated with 10, 2, 0.4, or 0.08 μg/ml of the respective mAb in thepresence of cross-linking antibody (5 μg/ml goat anti-human IgG Fc) for18 hours at 37° C. mAb 11 served as the isotype control. Cells wereharvested and washed after 18 hours and incubated with Annexin V-APC andPropidium Iodide for 15 minutes at 25° C. Cells were analyzed by flowcytometry for the presence of Annexin V positive/PI negative cells.

As can be seen in FIG. 10, the optimized humanized mAbs 1236, 1237, and1238 are as effective at inducing apoptosis as mAb 271(rituximab-sequence antibody).

Example 3 Further Optimization of mAb 1237

B-cell Chronic Lymphocytic Leukemia (B-CLL) cells express lower levelsof CD20 than NHL (Non-Hodgkins Lymphoma) cells. Testing showed thatoptimized mAb 1236, mAb 1237, and mAb 1238 demonstrate weak CDC of B-CLLcells. As a next step the mAb 1237 was selected and subjected tomutagenesis to screen for improvements in affinity and thus improvementsin the lysis of B-CLL cells. The affinity improvement strategy comprisedrandomizing specific positions in the V_(H) and the V_(K) domains,selecting mutant V_(H) domains with L373, selecting mutant V_(K) domainswith H1570, combining separate beneficial mutations in either V_(H) orV_(K), and combining mutant V_(H) with mutant V_(K) domains. The newmutants identified are shown in Table 3. TABLE 3 New mutants identified.Residue positions within the V_(H) and V_(K) domains are with referenceto the Kabat numbering system. mAb V_(H) V_(K) 1588 H1638 (H1570 S31F)L373 1589 H1639 (H1570 D56A) L373 1590 H1640 (H1570 D56L) L373 1652H1639 (H1570 D56A) L419 (L373 T92Q) 1692 H1670 (H1570 N101G) L373

The optimized mAb 1588 comprises the H1638 V_(H) domain (SEQ ID NO:15)(which is the H1570 V_(H) domain with an S→F substitution atKabat-numbering position 31 of H1570, which corresponds to residue 31 ofthe H1570 amino acid sequence set forth in SEQ ID NO:13) and the L373V_(L) domain (SEQ ID NO:10). This SF substitution results in anoptimized CDR1 (see SEQ ID NO:7) within the H1638 V_(H) domain.

The optimized mAb 1589 comprises the H1639 V_(H) domain (SEQ ID NO:16)(which is the H1570 V_(H) domain with a D→A substitution atKabat-numbering position 56 of H1570, which corresponds to residue 57 ofthe H1570 amino acid sequence set forth in SEQ ID NO:13) and the L373V_(L) domain (SEQ ID NO:10). This D→A substitution results in anoptimized CDR2 (see SEQ ID NO:5; encoded by SEQ ID NO:27) within theH1639 V_(H) domain.

The optimized mAb1590 comprises the H1640 V_(H) domain (SEQ ID NO:17)(which is the H1570 V_(H) domain with a D→L substitution atKabat-numbering position 56 of H1570, which corresponds to residue 57 ofthe H1570 amino acid sequence set forth in SEQ ID NO:13) and the L373V_(L) domain (SEQ ID NO:10). This D→L substitution results in anoptimized CDR2 (see SEQ ID NO:6) within the H1640 V_(H) domain.

The optimized mAb 1652 comprises the H1639 V_(H) domain (SEQ ID NO:16)and the L419 V_(L) domain (SEQ ID NO:11) (which is the L373 V_(L) domainwith a T→Q substitution at Kabat-numbering position 92 of L373, whichcorresponds to residue 91 of the L373 amino acid sequence set forth inSEQ ID NO:10). This T→Q substitution results in an optimized CDR3 (seeSEQ ID NO:8) within the L419 V_(L) domain.

The optimized mAb 1692 comprises the H1670 V_(H) domain (SEQ ID NO:18)(which is the H1570 V_(H) domain with an N→G substitution atKabat-numbering position 101 of H1570, which corresponds to residue 109of the H1570 amino acid sequence set forth in SEQ ID NO:13). This N→Gsubstitution results in an optimized CDR3 (see SEQ ID NO:4) within theH1670 V_(H) domain.

The amino acid sequences for the H1639 V_(H) domain (SEQ ID NO:16) andL373 V_(L) domain (SEQ ID NO:10) of the mAb 1589 are shown in FIG. 11,and the respective coding sequences are shown in FIG. 12; see also SEQID NO:22 (coding sequence for H1639) and SEQ ID NO:23 (coding sequencefor L373).

Example 4 Binding and Functional Characteristics of Additional OptimizedHumanized Murine Anti-CD20 Monoclonal Antibodies

The optimized humanized mAb 1237 of Example 1, and further optimizedhumanized mAbs 1588, 1589, 1590, 1652, and 1692 described in Example 3were assessed for their respective binding and functionalcharacteristics. For each assay, mAb 271, having the identical sequenceto rituximab (IDEC-C2B8; Rituxan®; IDEC Pharmaceuticals Corp., SanDiego, Calif.), served as the positive control.

Binding specificity was assessed by Flow Cytometry, which tabulates MeanFluorescence Intensities (MFI) of staining on CD20 positive (CD20⁺) andCD20 negative cell lines. Flow cytometry analysis demonstrated bindingof these optimized humanized anti-CD20 mAbs to CD20, as can be seen inTable 4. TABLE 4 Binding to CD20 membrane positive and negative celllines Flow Cytometry Results (Mean Fluorescence Intensities). Daudi EHEBHL60 K562 CHO CD20 CD20 CHO.CD20 CD20 CD20 Vector mAb Positive PositiveCD20 Positive Negative Negative CD20 Negative  271 941 130 81 6 3 4 12371373 161 106 8 9 6 1588 2068 338 140 9 6 4 1589 1029 345 140 8 6 5 15901627 429 135 8 7 6 1652 1422 529 135 6 6 5 1692 1671 450 137 7 4 5  11 68 5 7 4 6 (Negative Control)

Epitope conservation was assessed by examining the ability of the mAb2H7 to block binding. In this manner, mouse 2H7 (or Isotype Ig ascontrol) was added to EB1 cells, then candidate human mAbs andanti-human Ig Alexa 647 were added. Binding was detected by FMAT. Adecrease in the signal indicates blocking and common epitope usage.

As can be seen in FIG. 13, all tested antibodies recognized identical oroverlapping epitopes.

Complement dependent cytotoxicity (CDC) and antibody-dependent cellularcytotoxicity (ADCC) were assessed. For CDC, several assays were used.The first radioactive assay comprised loading target cells with ⁵¹Cr,200 μCi for 2 hours. Washed target cells (Daudi (NHL) and B-CLL lines)were incubated with titrated concentrations of mAb and human serum(complement source) for 4 hours. Controls included spontaneous andmaximum ⁵¹Cr release, mAb with cells alone, and serum with cells aloneat all concentrations. Supernatants were processed and released ⁵¹Crcounted with a gamma counter. Antibody specific CDC was determined bysubtracting out mAb and serum alone contributions to lysis. Results forDaudi and two B-CLL cell lines are shown in FIG. 14 and FIGS. 15A and15B, respectively. For Daudi cells, the optimized mAbs generallymediated greater CDC function than mAb 271 (rituximab-sequenceantibody), particularly at lower concentrations. For B-CLL cells, theoptimized mAbs showed increased CDC function over the mAb 271(rituximab-sequence antibody). CDC function was also assessed with anantibody off-rate assay. In this manner, target CD20⁺Daudi cells wereloaded with ⁵¹Cr, 200 μCi for 2 hours, and then washed. The washed Daudicells were incubated with 10 μg/ml of respective mAb for 15 minutes at25° C. Target Daudi cells were then washed thoroughly and incubated with6% human serum (complement source) for 45′ following 0, 1, 2, 4, and 6hour incubations at 37° C. Controls included spontaneous and maximum⁵¹Cr release, mAb with Daudi cells alone, and serum with Daudi cellsalone at all concentrations. Supernatents were then processed andreleased ⁵¹Cr counted via gamma counter. Antibody-specific CDC wasdetermined by subtracting out mAb and serum alone contributions tolysis.

As can be seen in FIG. 16, all optimized mAbs have significantly betterCDC functional activity than mAb 271 (rituximab-sequence antibody).

CDC function was also assessed with a non-radioactive (Alamar Blue™) CDCassay. In this manner, target cells (two each of NHL, B-CLL, and normalB cell lines) were washed with titrated concentrations of mAb and humanserum (complement source). Controls included target cells and antibodywithout human serum (spontaneous cell death), target cells and serumwithout antibody (background lysis), and wells of target cells with 10%SDS (or 5% Triton X100 in water) replacing the antibody and serum (formaximum cell death). Alamar Blue was added to each well 2 hours later,and fluorescence read after an overnight incubation. In this assay, morefluorescence indicates more live cells and less cytotoxicity due to CDC.

AlamarBlue™ is an oxidation-reduction indicator that fluoresces inresponse to metabolic activities in proliferating cells. AlamarBlue™ issimilar to tetrazolium salts (MTT), in that both can detect changes incell's metabolism, but AlamarBlue™ is less toxic and more sensitive thanMTT. Results obtained in the measurement of cell mediated cytotoxicitycomparing the use of AlamarBlue™ with ⁵¹Cr release assays indicate thatthe AamarBlue™ method is as specific as determination of ⁵¹Cr release.Results are shown in FIG. 17A (Daudi NHL cell line) and 17B (WIL2-S NHLcell line), FIG. 18A (EHEB B-CLL cell line) and 18B (Mec-1 B-CLL cellline), and FIG. 19A (SS BLCL “normal” B cells) and 19B (MeI K BLCL“normal” B cells).

The radioactive assay is a 4 hour assay while the non-radioactive assayis an 18-20 hour assay. The optimized mAb candidates usually gavegreater lysis in non-radioactive assays, as compared to controls. Thenon-radioactive assays may represent a more similar situation to thein-vivo setting, as the drug would be present at high concentrations formuch longer than 4 hours in an in-vivo setting.

To summarize the results, the CDC activity of the optimized mAbs is atleast equal to mAb 271 (rituximab-sequence antibody) on Daudi NHL cells.The optimized mAbs are better than mAb 271 in the off-rate CDC assay.The lower off-rate indicates that the affinity of the optimized MAbs ishigher than the affinity of mAb 271. Finally, the optimized mAbs arebetter than mAb 271 in lysing B-CLL cell lines and normal B celltargets.

ADCC activity was assayed by loading target cells (Daudi and B-CLL) with⁵¹Cr, 200 μCi for 2 hours, and following with a wash. The washed targetcells were incubated with titrated concentrations of respective mAbs andfreshly prepared PBMCs (NK source) at 50:1 for 4 hours. Controlsincluded spontaneous and maximum ⁵¹Cr release, mAb with target cellsalone, and PBMC with target cells alone at all concentrations.Supernatents were then processed and released ⁵¹Cr counted via gammacounter. Antibody-specific ADCC was determined by subtracting out PBMCcontribution to lysis.

As can be seen in FIG. 20 (Daudi NHL cells) and FIG. 21 (MEC-1 B-CLLcells), ADCC activity increased with increasing mAb concentration forall mAbs tested. The optimized mAbs had ADCC activity that is similar tothat observed for the control mAb 271 (rituximab-sequence antibody).

Apoptosis activity was also assessed using a direct apoptosis assay thatis CDC and ADCC independent. In this manner, Ramos cells (NHL) wereincubated with 2, 0.4, or 0.08 μg/ml of the respective optimized mAb orcontrol mAb 271 in the presence of cross-linking antibody (5 μg/ml goatanti-human IgG Fc) for 18 hours at 37° C. Cells were harvested andwashed after 18 hours and incubated with Annexin V-APC and PropidiumIodide for 15 minutes at 25° C. Cells were analyzed by flow cytometryfor the presence of Annexin V positive/PI negative cells.

As can be seen in FIG. 22, the optimized mAbs are as effective atinducing apoptosis as mAb 271 (rituximab-sequence antibody).

Cell killing activity was also assessed using a whole blood assay. Theprotocol was similar to CDC or ADCC assays, except whole blood is usedinstead of serum (CDC) or purified PBMCs (ADCC). In this assay, thelysis of target cells is due to cumulative activity of CDC, ADCC, andapoptosis.

Results of this whole blood assay are shown in FIG. 23 (Daudi NHLcells), FIG. 24A (EHEB B-CLL cells) and 24B (MEC-1 B-CLL cells), andFIG. 25 (MeI K “normal” B cells). As can be seen in these figures, theoptimized mAbs have equivalent or better lysis against these respectivetarget cells as mAb 271 (rituximab—sequence antibody).

Example 5 Administration of the Optimized Humanized Murine Anti-CD20Monoclonal Antibody 1589 Prolongs Survival in a Daudi Cell XenograftMouse Model

Daudi NHL cells are a human B cell lymphoma cell line that growsystematically in SCID mice. Growth of Daudi cells results in paralysisin the mice (Ghetie et al. (1990) Int J Cancer 45:481-485). Studies wereperformed to determine if the anti-CD20 antibodies of the inventioneffect the survival of mice with Daudi cell xenografts.

SCID mice were bred and maintained under pathogen-free conditions. DaudiNHL cells (8×10⁶) were injected intravenously into the tail vein ofthree groups of five SCID mice (15 mice total). On days 7 and 9post-tumor injection, the mice were injected by the same route with PBS,50 μg human IgG1 or 50 μg mAb 1589. Animals were observed three timesper week and sacrificed at the first signs of hind limb paralysis. Asshown in FIG. 26, the monoclonal antibody 1589 prolonged the survival ofmice with the human Daudi cell xenograft.

Example 6 Outline of Sequences and Antibody Design

The sequence identifiers for the various optimized CDRs, variable heavyand light domains comprising these optimized CDRs, coding sequences forthese elements, and variable heavy domain for the original humanizedanti-CD20 antibody are summarized below. Table 5 below outlines theanti-CD20 antibody design. SEQUENCE IDENTIFIERS:  1. CDR3 of H1569 -a.a.  2. CDR3 of H1570 - a.a.  3. CDR3 of H1571 - a.a.  4. CDR3 ofH1670 - a.a.  5. CDR2 of H1639 - a.a.  6. CDR2 of H1640 - a.a.  7. CDR1of H1638 - a.a.  8. CDR3 of L419 - a.a.  9. CDR2 of L373 - a.a. 10.L373 - a.a. 11. L419 - a.a. 12. H1569 - a.a. 13. H1570 - a.a. 14.H1571 - a.a. 15. H1638 - a.a. 16. H1639 - a.a. 17. H1640 - a.a. 18.H1670 - a.a. 19. H1569 - nt seq 20. H1570 - nt seq 21. H1571 - nt seq22. H1639 - nt seq 23. L373 - nt seq 24. CDR3 of H1569 - nt seq 25. CDR3of H1570 - nt seq 26. CDR3 of H1571 - nt seq 27. CDR2 of H1639 - nt seq28. CDR2 of L373 - nt seq 29. H1286 - a.a. 30. CDR3 of H1286 - a.a.

TABLE 5 Antibody design. ANTIBODY NAME V_(H) DOMAIN V_(L) DOMAIN Parenthumanized H1286 (SEQ ID NO: 29) L373 (SEQ ID NO: 10) murine anti-CD201236 H1569 (SEQ ID NO: 12) L373 (SEQ ID NO: 10) 1237 H1570 (SEQ ID NO:13) L373 (SEQ ID NO: 10) 1238 H1571 (SEQ ID NO: 14) L373 (SEQ ID NO: 10)1588 H1638 (SEQ ID NO: 15) L373 (SEQ ID NO: 10) 1589 H1639 (SEQ ID NO:16) L373 (SEQ ID NO: 10) 1590 H1640 (SEQ ID NO: 17) L373 (SEQ ID NO: 10)1593 H1570 (SEQ ID NO: 13) L419 (SEQ ID NO: 11) 1652 H1639 (SEQ ID NO:16) L419 (SEQ ID NO: 11) 1692 H1670 (SEQ ID NO: 18) L373 (SEQ ID NO: 10)

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims and listof embodiments disclosed herein. Although specific terms are employedherein, they are used in a generic and descriptive sense only and notfor purposes of limitation.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

1. An immunoglobulin that specifically binds CD20, wherein saidimmunoglobulin comprises at least one optimizedcomplementarity-determining (CDR), wherein said CDR comprises an aminoacid sequence selected from the group consisting of: a. the amino acidsequence set forth in SEQ ID NO:1; b. an amino acid sequence having atleast 85% sequence identity to the amino acid sequence set forth in SEQID NO:1, wherein said CDR comprises at least one residue selected fromthe group consisting of: i. the asparagine (Asn) residue at the positioncorresponding to residue 9 of SEQ ID NO:1; and ii. the asparagine (Asn)residue at the position corresponding to residue 12 of SEQ ID NO:1; c.the amino acid sequence set forth in SEQ ID NO:2; d. an amino acidsequence having at least 85% sequence identity to the amino acidsequence set forth in SEQ ID NO:2, wherein said CDR comprises at leastone residue selected from the group consisting of: i. the alanine (Ala)residue at the position corresponding to residue 5 of SEQ ID NO:2; ii.the asparagine (Asn) residue at the position corresponding to residue 9of SEQ ID NO:2; and iii. the asparagine (Asn) residue at the positioncorresponding to residue 12 of SEQ ID NO:2; e. the amino acid sequenceset forth in SEQ ID NO:3; f. an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:3,wherein said CDR comprises at least one residue selected from the groupconsisting of: i. the alanine (Ala) residue at the positioncorresponding to residue 5 of SEQ ID NO:3; ii. the asparagine (Asn)residue at the position corresponding to residue 9 of SEQ ID NO:3; andiii. the aspartic acid (Asp) residue at the position corresponding toresidue 12 of SEQ ID NO:3; g. the amino acid sequence set forth in SEQID NO:4; h. an amino acid sequence having at least 85% sequence identityto the amino acid sequence set forth in SEQ ID NO:4, wherein said CDRcomprises at least one residue selected from the group consisting of: i.the alanine (Ala) residue at the position corresponding to residue 5 ofSEQ ID NO:4; ii. the asparagine (Asn) residue at the positioncorresponding to residue 9 of SEQ ID NO:4; iii. the glycine (Gly)residue at the position corresponding to residue 11 of SEQ ID NO:4; andiv. the asparagine (Asn) residue at the position corresponding toresidue 12 of SEQ ID NO:4; j. the amino acid sequence set forth in SEQID NO:5; k. an amino acid sequence having at least 85% sequence identityto the amino acid sequence set forth in SEQ ID NO:5, wherein said CDRcomprises the alanine (Ala) residue at the position corresponding toresidue 8 of SEQ ID NO:5; l. the amino acid sequence set forth in SEQ IDNO:6; m. an amino acid sequence having at least 85% sequence identity tothe amino acid sequence set forth in SEQ ID NO:6, wherein said CDRcomprises the leucine (Leu) residue at the position corresponding toresidue 8 of SEQ ID NO:6; n. the amino acid sequence set forth in SEQ IDNO:7; o. an amino acid sequence having at least 85% sequence identity tothe amino acid sequence set forth in SEQ ID:7, wherein said CDRcomprises the phenylalanine (Phe) residue at the position correspondingto residue 7 of SEQ ID NO:7; p. the amino acid sequence set forth in SEQID NO:8; and q. an amino acid sequence having at least 85% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:8, whereinsaid CDR comprises the glutamine (Gln) residue at the positioncorresponding to residue 4 of SEQ ID NO:8.
 2. The immunoglobulin ofclaim 1, wherein said immunoglobulin comprises an immunoglobulin heavychain comprising a variable domain having at least one CDR selected fromthe group consisting of: a. a CDR1 comprising the amino acid sequenceset forth in SEQ ID NO:7; b. a CDR1 comprising an amino acid sequencehaving at least 85% sequence identity to the amino acid sequence setforth in SEQ ID:7, wherein said CDR1 comprises the phenylalanine (Phe)residue at the position corresponding to residue 7 of SEQ ID NO:7; c. aCDR2 comprising the amino acid sequence set forth in SEQ ID NO:5 or SEQID NO:6; d. a CDR2 comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID:5 orSEQ ID NO:6, wherein said CDR2 comprises the alanine (Ala) or leucine(Leu) residue at the position corresponding to residue 8 of SEQ ID NO:5or SEQ ID NO:6, respectively; e. a CDR3 comprising the amino acidsequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ IDNO:4; f. a CDR3 comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:1,wherein said CDR3 comprises at least one residue selected from the groupconsisting of: i. the asparagine (Asn) residue at the positioncorresponding to residue 9 of SEQ ID NO:1; and ii. the asparagine (Asn)residue at the position corresponding to residue 12 of SEQ ID NO:1; g. aCDR3 comprising an amino acid sequence having at least 85% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:2, whereinsaid CDR3 comprises at least one residue selected from the groupconsisting of: i. the alanine (Ala) residue at the positioncorresponding to residue 5 of SEQ ID NO:2 ii. the asparagine (Asn)residue at the position corresponding to residue 9 of SEQ ID NO:2; andiii. the asparagine (Asn) residue at the position corresponding toresidue 12 of SEQ ID NO:2; h. a CDR3 comprising an amino acid sequencehaving at least 85% sequence identity to the amino acid sequence setforth in SEQ ID NO:3, wherein said CDR3 comprises at least one residueselected from the group consisting of: i. the alanine (Ala) residue atthe position corresponding to residue 5 of SEQ ID NO:3; ii. theasparagine (Asn) residue at the position corresponding to residue 9 ofSEQ ID NO:3; and iii. the aspartic acid (Asp) residue at the positioncorresponding to residue 12 of SEQ ID NO:3; and i. a CDR3 comprising anamino acid sequence having at least 85% sequence identity to the aminoacid sequence set forth in SEQ ID NO:4, wherein said CDR3 comprises atleast one residue selected from the group consisting of: i. the alanine(Ala) residue at the position corresponding to residue 5 of SEQ ID NO:4;ii. the asparagine (Asn) residue at the position corresponding toresidue 9 of SEQ ID NO:4; iii. the glycine (Gly) residue at the positioncorresponding to residue 11 of SEQ ID NO:4; and iv. the asparagine (Asn)residue at the position corresponding to residue 12 of SEQ ID NO:4. 3.The immunoglobulin of claim 2, wherein said immunoglobulin comprises animmunoglobulin light chain comprising a variable domain having at leastone CDR selected from the group consisting of: a. a CDR3 comprising theamino acid sequence set forth in SEQ ID NO: 8; b. a CDR3 comprising anamino acid sequence having at least 85% sequence identity to the aminoacid sequence set forth in SEQ ID NO:8, wherein said CDR3 comprises theglutamine (Gln) residue at the position corresponding to residue 4 ofSEQ ID NO:8; and c. a CDR2 comprising the amino acid sequence set forthin SEQ ID NO:
 9. 4. The immunoglobulin of claim 3, wherein the variabledomain of said light chain comprises the CDR2 set forth in SEQ ID NO:9and a CDR3 selected from the group consisting of: a. a CDR3 comprisingthe amino acid sequence set forth in SEQ ID NO:8; and b. a CDR3comprising an amino acid sequence having at least 85% sequence identityto the amino acid sequence set forth in SEQ ID NO:8, wherein said CDR3comprises the glutamine (Gln) residue at the position corresponding toresidue 4 of SEQ ID NO:8.
 5. The immunoglobulin of claim 1, wherein saidimmunoglobulin comprises an immunoglobulin light chain comprising avariable domain having at least one CDR selected from the groupconsisting of: a. a CDR3 comprising the amino acid sequence set forth inSEQ ID NO: 8; and b. a CDR3 comprising an amino acid sequence having atleast 85% sequence identity to the amino acid sequence set forth in SEQID NO:8, wherein said CDR3 comprises the glutamine (Gln) residue at theposition corresponding to residue 4 of SEQ ID NO:8.
 6. Theimmunoglobulin of claim 5, wherein the variable domain of said lightchain further comprises the CDR2 set forth in SEQ ID NO:9.
 7. Theimmunoglobulin of claim 1, wherein said immunoglobulin is an IgG1 kappaimmunoglobulin.
 8. The immunoglobulin of claim 7, wherein said IgG1kappa immunoglobulin comprises a human IgG1 constant region within aheavy chain of said immunoglobulin and a human kappa constant regionwithin a light chain of said immunoglobulin.
 9. The immunoglobulin ofclaim 8, wherein said immunoglobulin comprises fully or partially humanframework regions within the variable domain of said heavy chain andwithin the variable domain of said light chain.
 10. The immunoglobulinof claim 8, wherein said immunoglobulin comprises murine frameworkregions within the variable domain of said heavy chain and within thevariable domain of said light chain.
 11. The immunoglobulin of claim 1,wherein said immunoglobulin exhibits increased complement-dependentcytotoxicity (CDC) as compared to rituximab.
 12. The immunoglobulin ofclaim 1, further comprising a heterologous polypeptide fused thereto.13. The immunoglobulin of claim 1, wherein said immunoglobulin isconjugated to an agent selected from the group consisting of atherapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus,a lipid, a biological response modifier, a pharmaceutical agent, andPEG.
 14. A composition comprising the immunoglobulin of claim
 1. 15. Thecomposition of claim 14, further comprising a pharmaceuticallyacceptable carrier.
 16. A method for treating a disease in a subject,wherein the disease is a cancer, an autoimmune disease, or aninflammatory disease, comprising administering to said subject aneffective amount of a composition comprising the immunoglobulin ofclaim
 1. 17. The method of claim 16, wherein said cancer is selectedfrom the group consisting of a non-Hodgkins lymphoma, chroniclymphocytic leukemia, multiple myeloma, B cell lymphoma, high-grade Bcell lymphoma, intermediate-grade B cell lymphoma, low-grade B celllymphoma, B cell acute lymphoblastic leukemia, myeloblastic leukemia,and Hodgkin's disease.
 18. The method of claim 16, wherein theautoimmune disease or inflammatory disease is selected from the groupconsisting of systemic lupus erythematosus (SLE), discoid lupus, lupusnephritis, sarcoidosis, juvenile arthritis, rheumatoid arthritis,psoriatic arthritis, Reiter's syndrome, ankylosing spondylitis, goutyarthritis, rejection of an organ or tissue transplant, graft versus hostdisease, multiple sclerosis, hyper IgE syndrome, polyarteritis nodosa,primary biliary cirrhosis, inflammatory bowel disease, Crohn's disease,celiac's disease (gluten-sensitive enteropathy), autoimmune hepatitis,pernicious anemia, autoimmune hemolytic anemia, psoriasis, scleroderma,myasthenia gravis, autoimmune thrombocytopenic purpura, autoimmunethyroiditis, Grave's disease, Hashimoto's thyroiditis, immune complexdisease, chronic fatigue immune dysfunction syndrome (CFIDS),polymyositis and dermatomyositis, cryoglobulinemia, thrombolysis,cardiomyopathy, pemphigus vulgaris, pulmonary interstitial fibrosis,sarcoidosis, Type I and Type II diabetes mellitus, type 1, 2, 3, and 4delayed-type hypersensitivity, allergy or allergic disorders, asthma,Churg-Strauss syndrome (allergic granulomatosis), atopic dermatitis,allergic and irritant contact dermatitis, urtecaria, IgE-mediatedallergy, atherosclerosis, vasculitis, idiopathic inflammatorymyopathies, hemolytic disease, Alzheimer's disease, and chronicinflammatory demyelinating polyneuropathy.
 19. A method for inhibitinggrowth or differentiation of a normal human B cell or cancer cells of Bcell lineage, comprising contacting said B cell or said cancer cellswith an effective amount of the immunoglobulin according to claim
 1. 20.The method of claim 19, wherein the cancer cells are from a cancerselected from the group consisting of non-Hodgkins lymphoma, chroniclymphocytic leukemia, multiple myeloma, B cell lymphoma, high-grade Bcell lymphoma, intermediate-grade B cell lymphoma, low-grade B celllymphoma, B cell acute lymphoblastic leukemia, myeloblastic leukemia,and Hodgkin's disease.
 21. An immunoglobulin that specifically bindsCD20, wherein said immunoglobulin comprises a variable heavy (V_(H))domain, wherein said V_(H) domain is selected from the group consistingof: a. a V_(H) domain comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOS:12-18; b. a V_(H) domain comprisingan amino acid sequence having at least 90% sequence identity to any oneof the amino acid sequences set forth in SEQ ID NOS: 12-18; c. a V_(H)domain comprising an amino acid sequence having at least 85% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:12, whereinsaid V_(H) domain comprises at least one residue selected from the groupconsisting of: i. the asparagine (Asn) residue at the positioncorresponding to residue 107 of SEQ ID NO:12; and ii. the asparagine(Asn) residue at the position corresponding to residue 110 of SEQ IDNO:12; d. a V_(H) domain comprising an amino acid sequence having atleast 85% sequence identity to the amino acid sequence set forth in SEQID NO:13, wherein said V_(H) domain comprises at least one residueselected from the group consisting of: i. the alanine (Ala) residue atthe position corresponding to residue 103 of SEQ ID NO:13; ii. theasparagine (Asn) residue at the position corresponding to residue 107 ofSEQ ID NO:13; and iii. the asparagine (Asn) residue at the positioncorresponding to residue 110 of SEQ ID NO:13; e. a V_(H) domaincomprising an amino acid sequence having at least 85% sequence identityto the amino acid sequence set forth in SEQ ID NO:14, wherein said V_(H)domain comprises at least one residue selected from the group consistingof: i. the alanine (Ala) residue at the position corresponding toresidue 103 of SEQ ID NO:14; ii. the asparagine (Asn) residue at theposition corresponding to residue 107 of SEQ ID NO:14; and iii. theaspartic acid (Asp) residue at the position corresponding to residue 110of SEQ ID NO:14; f. a V_(H) domain comprising an amino acid sequencehaving at least 85% sequence identity to the amino acid sequence setforth in SEQ ID NO:15, wherein said V_(H) domain comprises at least oneresidue selected from the group consisting of: i. the phenylalanine(Phe) residue at the position corresponding to residue 31 of SEQ IDNO:15; ii. the alanine (Ala) residue at the position corresponding toresidue 103 of SEQ ID NO:15; iii. the asparagine (Asn) residue at theposition corresponding to residue 107 of SEQ ID NO:15; and iv. theasparagine (Asn) residue at the position corresponding to residue 110 ofSEQ ID NO:15; g. a V_(H) domain comprising an amino acid sequence havingat least 85% sequence identity to the amino acid sequence set forth inSEQ ID NO:16, wherein said V_(H) domain comprises at least one residueselected from the group consisting of: i. the alanine (Ala) residue atthe position corresponding to residue 57 of SEQ ID NO:16; ii. thealanine (Ala) residue at the position corresponding to residue 103 ofSEQ ID NO:16; iii. the asparagine (Asn) residue at the positioncorresponding to residue 107 of SEQ ID NO:16; and iv. the asparagine(Asn) residue at the position corresponding to residue 110 of SEQ IDNO:16; h. a V_(H) domain comprising an amino acid sequence having atleast 85% sequence identity to the amino acid sequence set forth in SEQID NO:17, wherein said V_(H) domain comprises at least one residueselected from the group consisting of: i. the leucine (Leu) residue atthe position corresponding to residue 57 of SEQ ID NO:17; ii. thealanine (Ala) residue at the position corresponding to residue 103 ofSEQ ID NO:17; iii. the asparagine (Asn) residue at the positioncorresponding to residue 107 of SEQ ID NO:17; and iv. the asparagine(Asn) residue at the position corresponding to residue 110 of SEQ IDNO:17; and i. a V_(H) domain comprising an amino acid sequence having atleast 85% sequence identity to the amino acid sequence set forth in SEQID NO:18, wherein said V_(H) domain comprises at least one residueselected from the group consisting of: i. the alanine (Ala) residue atthe position corresponding to residue 103 of SEQ ID NO:18; ii. theasparagine (Asn) residue at the position corresponding to residue 107 ofSEQ ID NO:18; iii. the glycine (Gly) residue at the positioncorresponding to residue 109 of SEQ ID NO:18; and iv. the asparagine(Asn) residue at the position corresponding to residue 110 of SEQ IDNO:18.
 22. The immunoglobulin of claim 21, wherein said immunoglobulincomprises a variable light (V_(L)) domain, wherein said V_(L) domain isselected from the group consisting of: a. a V_(L) domain comprising theamino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:1; b. a V_(L)domain comprising an amino acid sequence having at least 90% sequenceidentity to the sequence set forth in SEQ ID NO:10 or SEQ ID NO:11. c. aV_(L) domain comprising an amino acid sequence having at least 85%sequence identity to the sequence set forth in SEQ ID NO:10, whereinsaid V_(L) domain comprises the complementarity-determining region 2(CDR2) set forth in SEQ ID NO:9; d. a V_(L) domain comprising an aminoacid sequence having at least 85% sequence identity to the sequence setforth in SEQ ID NO:11, wherein said V_(L) domain comprises at least oneof: i. the complementarity-determining region 2 (CDR2) set forth in SEQID NO:9; and ii. the glutamine (Gln) residue at the positioncorresponding to residue 91 of SEQ ID NO:11.
 23. The immunoglobulin ofclaim 22, wherein said V_(H) domain comprises the amino acid sequenceset forth in SEQ ID NO:16 and said V_(L) domain comprises the amino acidsequence set forth in SEQ ID NO:10.
 24. An immunoglobulin thatspecifically binds CD20, wherein said immunoglobulin comprises avariable light (V_(L)) domain, wherein said V_(L) domain is selectedfrom the group consisting of: a. a V_(L) domain comprising the aminoacid sequence set forth in SEQ ID NO:10 or SEQ ID NO:11; b. a V_(L)domain comprising an amino acid sequence having at least 90% sequenceidentity to the sequence set forth in SEQ ID NO:10 or SEQ ID NO:11; c. aV_(L) domain comprising an amino acid sequence having at least 85%sequence identity to the sequence set forth in SEQ ID NO:10, whereinsaid V_(L) domain comprises the complementarity-determining region 2(CDR2) set forth in SEQ ID NO:9; d. a V_(L) domain comprising an aminoacid sequence having at least 85% sequence identity to the sequence setforth in SEQ ID NO:11, wherein said V_(L) domain comprises at least oneof: i. the complementarity-determining region 2 (CDR2) set forth in SEQID NO:9; and ii. the glutamine (Gln) residue at the positioncorresponding to residue 91 of SEQ ID NO:11.
 25. An isolatedpolynucleotide comprising a nucleic acid encoding a variable heavy(V_(H)) domain of an immunoglobulin heavy chain, wherein said V_(H)domain is selected from the group consisting of: a. a V_(H) domaincomprising an amino acid sequence comprising at least one optimizedcomplementarity-determining region (CDR), wherein said CDR comprises anamino acid sequence selected from the group consisting of SEQ IDNOS:1-7; b. a V_(H) domain comprising at least one CDR selected from thegroup consisting of: i. a CDR1 comprising the amino acid sequence setforth in SEQ ID NO:7; ii. a CDR1 comprising an amino acid sequencehaving at least 85% sequence identity to the amino acid sequence setforth in SEQ ID:7, wherein said CDR1 comprises the phenylalanine (Phe)residue at the position corresponding to residue 7 of SEQ ID NO:7; iii.a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5 orSEQ ID NO:6; iv. a CDR2 comprising an amino acid sequence having atleast 85% sequence identity to the amino acid sequence set forth in SEQID:5 or SEQ ID NO:6, wherein said CDR2 comprises the alanine (Ala) orleucine (Leu) residue at the position corresponding to residue 8 of SEQID NO:5 or SEQ ID NO:6, respectively; v. a CDR3 comprising the aminoacid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQID NO:4; vi. a CDR3 comprising an amino acid sequence having at least85% sequence identity to the amino acid sequence set forth in SEQ IDNO:1, wherein said CDR3 comprises at least one residue selected from thegroup consisting of:
 1. the asparagine (Asn) residue at the positioncorresponding to residue 9 of SEQ ID NO:1; and
 2. the asparagine (Asn)residue at the position corresponding to residue 12 of SEQ ID NO:1; vii.a CDR3 comprising an amino acid sequence having at least 85% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:2, whereinsaid CDR3 comprises at least one residue selected from the groupconsisting of:
 1. the alanine (Ala) residue at the positioncorresponding to residue 5 of SEQ ID NO:2;
 2. the asparagine (Asn)residue at the position corresponding to residue 9 of SEQ ID NO:2; and3. the asparagine (Asn) residue at the position corresponding to residue12 of SEQ ID NO:2; viii. a CDR3 comprising an amino acid sequence havingat least 85% sequence identity to the amino acid sequence set forth inSEQ ID NO:3, wherein said CDR3 comprises at least one residue selectedfrom the group consisting of:
 1. the alanine (Ala) residue at theposition corresponding to residue 5 of SEQ ID NO:3;
 2. the asparagine(Asn) residue at the position corresponding to residue 9 of SEQ ID NO:3;and
 3. the aspartic acid (Asp) residue at the position corresponding toresidue 12 of SEQ ID NO:3; and ix. a CDR3 comprising an amino acidsequence having at least 85% sequence identity to the amino acidsequence set forth in SEQ ID NO:4, wherein said CDR3 comprises at leastone residue selected from the group consisting of:
 1. the alanine (Ala)residue at the position corresponding to residue 5 of SEQ ID NO:4; 2.the asparagine (Asn) residue at the position corresponding to residue 9of SEQ ID NO:4;
 3. the glycine (Gly) residue at the positioncorresponding to residue 11 of SEQ ID NO:4; and
 4. the asparagine (Asn)residue at the position corresponding to residue 12 of SEQ ID NO:4; c. aV_(H) domain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOS:12-18; d. a V_(H) domain comprising an aminoacid sequence having at least 90% sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NOS:12-18; e. aV_(H) domain comprising an amino acid sequence having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO:12,wherein said V_(H) domain comprises at least one residue selected fromthe group consisting of: i. the asparagine (Asn) residue at the positioncorresponding to residue 107 of SEQ ID NO:12; and ii. the asparagine(Asn) residue at the position corresponding to residue 110 of SEQ IDNO:12; f. a V_(H) domain comprising an amino acid sequence having atleast 85% sequence identity to the amino acid sequence set forth in SEQID NO:13, wherein said V_(H) domain comprises at least one residueselected from the group consisting of: i. the alanine (Ala) residue atthe position corresponding to residue 103 of SEQ ID NO:13; ii. theasparagine (Asn) residue at the position corresponding to residue 107 ofSEQ ID NO:13; and iii. the asparagine (Asn) residue at the positioncorresponding to residue 110 of SEQ ID NO:13; g. a V_(H) domaincomprising an amino acid sequence having at least 85% sequence identityto the amino acid sequence set forth in SEQ ID NO:14, wherein said V_(H)domain comprises at least one residue selected from the group consistingof: i. the alanine (Ala) residue at the position corresponding toresidue 103 of SEQ ID NO:14; ii. the asparagine (Asn) residue at theposition corresponding to residue 107 of SEQ ID NO:14; and iii. theaspartic acid (Asp) residue at the position corresponding to residue 110of SEQ ID NO:14; h. a V_(H) domain comprising an amino acid sequencehaving at least 85% sequence identity to the amino acid sequence setforth in SEQ ID NO:15, wherein said V_(H) domain comprises at least oneresidue selected from the group consisting of: i. the phenylalanine(Phe) residue at the position corresponding to residue 31 of SEQ IDNO:15; ii. the alanine (Ala) residue at the position corresponding toresidue 103 of SEQ ID NO:15; iii. the asparagine (Asn) residue at theposition corresponding to residue 107 of SEQ ID NO:15; and iv. theasparagine (Asn) residue at the position corresponding to residue 110 ofSEQ ID NO:15; i. a V_(H) domain comprising an amino acid sequence havingat least 85% sequence identity to the amino acid sequence set forth inSEQ ID NO:16, wherein said V_(H) domain comprises at least one residueselected from the group consisting of: i. the alanine (Ala) residue atthe position corresponding to residue 57 of SEQ ID NO:16; ii. thealanine (Ala) residue at the position corresponding to residue 103 ofSEQ ID NO:16; iii. the asparagine (Asn) residue at the positioncorresponding to residue 107 of SEQ ID NO:16; and iv. the asparagine(Asn) residue at the position corresponding to residue 110 of SEQ IDNO:16; j. a V_(H) domain comprising an amino acid sequence having atleast 85% sequence identity to the amino acid sequence set forth in SEQID NO:17, wherein said V_(H) domain comprises at least one residueselected from the group consisting of: i. the leucine (Leu) residue atthe position corresponding to residue 57 of SEQ ID NO:17; ii. thealanine (Ala) residue at the position corresponding to residue 103 ofSEQ ID NO:17; iii. the asparagine (Asn) residue at the positioncorresponding to residue 107 of SEQ ID NO:17; and iv. the asparagine(Asn) residue at the position corresponding to residue 110 of SEQ IDNO:17; and k. a V_(H) domain comprising an amino acid sequence having atleast 85% sequence identity to the amino acid sequence set forth in SEQID NO:18, wherein said V_(H) domain comprises at least one residueselected from the group consisting of: i. the alanine (Ala) residue atthe position corresponding to residue 103 of SEQ ID NO:18; ii. theasparagine (Asn) residue at the position corresponding to residue 107 ofSEQ ID NO:18; iii. the glycine (Gly) residue at the positioncorresponding to residue 109 of SEQ ID NO:18; and iv. the asparagine(Asn) residue at the position corresponding to residue 110 of SEQ IDNO:18.
 26. The isolated polynucleotide of claim 25, wherein said V_(H)domain is encoded by a nucleic acid comprising a sequence selected fromthe group consisting of SEQ ID NOS:19-22.
 27. The isolatedpolynucleotide of claim 25, further comprising vector sequence.
 28. Theisolated polynucleotide of claim 27, further comprising a nucleic acidencoding a variable light (V_(L)) domain of an immunoglobulin lightchain.
 29. The isolated polynucleotide of claim 28, wherein said V_(L)domain is selected from the group consisting of: a. a V_(L) domaincomprising at least one complementarity-determining (CDR), wherein saidCDR is selected from the group consisting of: i. a CDR comprising theamino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:9; ii. a CDR3comprising the amino acid sequence set forth in SEQ ID NO: 8; iii. aCDR3 comprising an amino acid sequence having at least 85% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:8, whereinsaid CDR3 comprises the glutamine (Gln) residue at the positioncorresponding to residue 4 of SEQ ID NO:8; and iv. a CDR2 comprising theamino acid sequence set forth in SEQ ID NO: 9; b. a V_(L) domaincomprising the amino acid sequence set forth in SEQ ID NO:10 or SEQ IDNO:1; c. a V_(L) domain comprising an amino acid sequence having atleast 90% sequence identity to the sequence set forth in SEQ ID NO:10 orSEQ ID NO:1; d. a V_(L) domain comprising an amino acid sequence havingat least 85% sequence identity to the sequence set forth in SEQ IDNO:10, wherein said V_(L) domain comprises thecomplementarity-determining region 2 (CDR2) set forth in SEQ ID NO:9; e.a V_(L) domain comprising an amino acid sequence having at least 85%sequence identity to the sequence set forth in SEQ ID NO:11, whereinsaid V_(L) domain comprises at least one of: i. thecomplementarity-determining region 2 (CDR2) set forth in SEQ ID NO:9;and ii. the glutamine (Gln) residue at the position corresponding toresidue 91 of SEQ ID NO:11.
 30. The isolated polynucleotide of claim 28,wherein said nucleic acid encoding said V_(H) domain and said nucleicacid encoding said V_(L) domain are fused in frame, are co-transcribedfrom a single promoter operably associated therewith, and arecotranslated into a single-chain antibody or antigen-binding fragmentthereof.
 31. The isolated polynucleotide of claim 28, wherein saidnucleic acid encoding said V_(H) domain and said nucleic acid encodingsaid V_(L) domain are co-transcribed from a single promoter operablyassociated therewith, but are separately translated.
 32. The isolatedpolynucleotide of claim 31, further comprising an IRES sequence disposedbetween said nucleic acid encoding said V_(H) domain and said nucleicacid encoding said V_(L) domain.
 33. The isolated polynucleotide ofclaim 28, wherein said nucleic acid encoding said V_(H) domain and saidnucleic acid encoding said V_(L) domain are separately transcribed, eachbeing operably associated with a separate promoter.
 34. The isolatedpolynucleotide of claim 33, wherein said separate promoters are copiesof the same promoter.
 35. The isolated polynucleotide of claim 33,wherein said separate promoters are non-identical.
 36. The isolatedpolynucleotide of claim 25, wherein said polynucleotide is within a hostcell.
 37. A method of producing an immunoglobulin that specificallybinds to CD20, comprising culturing the host cell of claim 36, andrecovering said immunoglobulin.
 38. An isolated polynucleotidecomprising a nucleic acid encoding a variable light (V_(L)) domain of animmunoglobulin light chain, wherein said V_(L) domain is selected fromthe group consisting of: a. a V_(L) domain comprising at least onecomplementarity-determining (CDR), wherein said CDR is selected from thegroup consisting of: i. a CDR3 comprising the amino acid sequence setforth in SEQ ID NO:8; and ii. a CDR3 comprising an amino acid sequencehaving at least 85% sequence identity to the amino acid sequence setforth in SEQ ID NO:8, wherein said CDR3 comprises the glutamine (Gln)residue at the position corresponding to residue 4 of SEQ ID NO:8; b. aV_(L) domain comprising the amino acid sequence set forth in SEQ IDNO:10 or SEQ ID NO:11; c. a V_(L) domain comprising an amino acidsequence having at least 90% sequence identity to the sequence set forthin SEQ ID NO:10 or SEQ ID NO:11; d. a V_(L) domain comprising an aminoacid sequence having at least 85% sequence identity to the sequence setforth in SEQ ID NO:10, wherein said V_(L) domain comprises thecomplementarity-determining region 2 (CDR2) set forth in SEQ ID NO:9;and e. a V_(L) domain comprising an amino acid sequence having at least85% sequence identity to the sequence set forth in SEQ ID NO:11, whereinsaid V_(L) domain comprises at least one of: i. thecomplementarity-determining region 2 (CDR2) set forth in SEQ ID NO:9;and ii. the glutamine (Gln) residue at the position corresponding toresidue 91 of SEQ ID NO:11.
 39. The isolated polynucleotide of claim 38,further comprising vector sequence.
 40. The isolated polynucleotide ofclaim 39, further comprising a nucleic acid encoding a variable heavy(V_(H)) domain of an immunoglobulin heavy chain.
 41. The isolatedpolynucleotide of claim 38, wherein said polynucleotide is within a hostcell.
 42. A method of producing an immunoglobulin that specificallybinds to CD20, comprising culturing the host cell of claim 41, andrecovering said immunoglobulin.